bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Cytochrome
c
Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of
Azospirillum lipoferum
Authors: Alexandre Gladys , Bally René , Taylor Barry L. , Zhulin Igor B. 
Published: December 2019

ABSTRACT

Laccase, a
p
-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium,
Azospirillum lipoferum
. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome
c
oxidase of the
aa
3
type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the
bc
1
complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome
c
oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.
Outer Membrane Lipoprotein
e
(P4) of
Haemophilus influenzae
Is a Novel Phosphomonoesterase
Authors: Reilly Thomas J. , Chance Deborah L. , Smith Arnold L. 
Published: December 2019

ABSTRACT

Haemophilus influenzae
exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable
H. influenzae
strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein
e
(P4) within the
H. influenzae
sequence database.
Escherichia coli
DH5α transformed with plasmids containing the
H. influenzae hel
gene, which encodes lipoprotein
e
(P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated ∼28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase.
Deletion of the
pyc
Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit
Authors: Pérez-Redondo Rosario , Rodríguez-García Antonio , Martín Juan F. , Liras Paloma 
Published: December 2019

ABSTRACT

The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (
pyc
, for pyruvate converting) located upstream of the
bls
gene in the clavulanic acid gene cluster of
Streptomyces clavuligerus
encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the
pyc
gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the
pyc
gene with the
aph
gene did not cause isoleucine-valine auxotrophy in the mutant. The
pyc
replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the
pyc
gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing
d
-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The
pyc
replacement mutant overproduces cephamycin.
A RecG-Independent Nonconservative Branch Migration Mechanism inEscherichia coliRecombination
Authors: Friedman-Ohana Rachel , Karunker Iris , Cohen Amikam 
Published: December 2019

ABSTRACTTo gain insight regarding the mechanisms that extend heteroduplex joints inEscherichia colirecombination, we investigated the effect ofrecGandruvgenotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and inruvmutants. However, inrecGmutants heteroduplexes were generated almost exclusively by pairing the invasive 3′-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect ofrecGandrecJorxonAmutations on recombination proficiency and the inhibitory effect ofrecJandxonAactivities on heteroduplex formation by the 5′-ending strands are consistent with this proposal.
Direct Selection of IS
903
Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of
Actinobacillus actinomycetemcomitans
Authors: Thomson Valeri J. , Bhattacharjee Mrinal K. , Fine Daniel H. , Derbyshire Keith M. , Figurski David H. 
Published: December 2019

ABSTRACT

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS
903
-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS
903φkan
, carries a cryptic
kan
gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS
903φkan
was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS
903φkan
insertion mutants were readily selected in
Escherichia coli
by their resistance to kanamycin. We used IS
903φkan
to isolate three catalase-deficient mutants of the periodontal pathogen
Actinobacillus actinomycetemcomitans
from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS
903φkan
insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated
katA
) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of
Haemophilus influenzae
. Our results demonstrate the utility of the IS
903φkan
mutagenesis system for the study of
A. actinomycetemcomitans
. Because IS
903φkan
is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
Regulation of Sigma 54-Dependent Transcription by Core Promoter Sequences: Role of −12 Region Nucleotides
Authors: Wang Lei , Guo Yuli , Gralla Jay D. 
Published: December 2019

ABSTRACT
The tetranucleotide core recognition sequence (TTGC) of the sigma 54 promoter −12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo and in vitro. Deregulated promoters were identified, implying that this core element can mediate the response to enhancer-binding proteins. These promoters had in common a substitution at position −12 (consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were identified. Their analysis indicated that positions −13 (consensus G) and −15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the −13 and −15 positions contribute to closed-complex formation, whereas the −12 position has a stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that the −12 region core contains specific subsequences that direct the diverse RNA polymerase interactions required both to produce RNA and to restrict this RNA synthesis in the absence of activation.
IS
1630
of
Mycoplasma fermentans
, a Novel IS
30
-Type Insertion Element That Targets and Duplicates Inverted Repeats of Variable Length and Sequence during Insertion
Authors: Calcutt Michael J. , Lavrrar Jennifer L. , Wise Kim S. 
Published: December 2019

ABSTRACT

A new insertion sequence (IS) of
Mycoplasma fermentans
is described. This element, designated IS
1630
, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS
30
family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS
30
. Multiple copies of IS
1630
are present in the genomes of at least two
M. fermentans
strains. Characterization and comparison of nine copies of the element revealed that IS
1630
exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS
1630
was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS
30
-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS
1630
vary between 19 and 26 bp. With the exception of two recently reported IS
4
-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037–1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319–1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS
1630
-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS
30
is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS
1630
appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence IS
Mi
1
was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS
3
family members than had previously been recognized. A potential site for programmed translational frameshifting in IS
Mi
1
was also identified.
Evidence that Expression of the
Vibrio vulnificus
Hemolysin Gene Is Dependent on Cyclic AMP and Cyclic AMP Receptor Protein
Authors: Bang Young Bae , Lee Shee Eun , Rhee Joon Haeng , Choi Sang Ho 
Published: December 2019

ABSTRACT

Glucose repressed hemolysin production in
Vibrio vulnificus
. Promoter activity of the hemolysin gene,
vvh
, assessed with a
vvh-luxCDABE
transcriptional fusion, required cyclic AMP (cAMP) and cAMP receptor protein (CRP) in
Escherichia coli
. Hemolysin production in
V. vulnificus
increased after the addition of cAMP and was undetectable in a putative
crp
mutant, suggesting that
vvh
is also regulated by cAMP-CRP in
V. vulnificus
.
Cloning and Characterization of a Gene (
mspA
) Encoding the Major Sheath Protein of
Treponema maltophilum
ATCC 51939
T
Authors: Heuner Klaus , Choi Bong-Kyu , Schade Rüdiger , Moter Annette , Otto Albrecht , Göbel Ulf B. 
Published: December 2019

ABSTRACT

The major sheath protein-encoding gene (
mspA
) of the oral spirochete
Treponema maltophilum
ATCC 51939
T
was cloned by screening a genomic library with an anti-outer membrane fraction antibody. The
mspA
gene encodes a precursor protein of 575 amino acids with a predicted molecular mass of 62.3 kDa, including a signal peptide of 19 amino acids. The native MspA formed a heat-modifiable, detergent- and trypsin-stable complex which is associated with the outer membrane. Hybridization with an
mspA
-specific probe showed no cross-reactivity with the
msp
gene from
Treponema denticola
.
Molecular Characterization of the
Lactococcus lactis ptsHI
Operon and Analysis of the Regulatory Role of HPr
Authors: Luesink Evert J. , Beumer Christel M. A. , Kuipers Oscar P. , De Vos Willem M. 
Published: December 2019

ABSTRACT

The
Lactococcus lactis ptsH
and
ptsI
genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of the
ptsHI
operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containing
ptsH
and a second of 2.0 kb containing both
ptsH
and
ptsI
. Disruption of the
ptsH
and
ptsI
genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in
L. lactis
. Complementation of the
ptsH
and
ptsI
mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the
gal
operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and
l
-lactate dehydrogenase genes.
Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability
Authors: Samartzidou Hrissi , Delcour Anne H. 
Published: December 2019

ABSTRACT

The permeability of the outer membrane of
Escherichia coli
to hydrophilic compounds is controlled by porin channels. Electrophysiological experiments showed that polyamines inhibit ionic flux through cationic porins when applied to either side of the membrane. Externally added polyamines, such as cadaverine, decrease porin-mediated fluxes of β-lactam antibiotics in live cells. Here we tested the effects of endogenously expressed cadaverine on the rate of permeation of cephaloridine through porins, by manipulating in a pH-independent way the expression of the
cadBA
operon, which encodes proteins involved in the decarboxylation of lysine to cadaverine and in cadaverine excretion. We report that increased levels of excreted cadaverine correlate with a decreased outer membrane permeability to cephaloridine, without any change in porin expression. Cadaverine appears to promote a sustained inhibition of porins, since the effect remains even after removal of the exogenously added or excreted polyamine. The cadaverine-induced inhibition is sufficient to provide cells with some resistance to ampicillin but not to hydrophobic antibiotics. Finally, the mere expression of
cadC
, in the absence of cadaverine production, leads to a reduction in the amounts of OmpF and OmpC proteins, which suggests a novel mechanism for the environmental control of porin expression. The results presented here support the notion that polyamines can act as endogenous modulators of outer membrane permeability, possibly as part of an adaptive response to acidic conditions.
Immunochemical Analysis of UMP Kinase from
Escherichia coli
Authors: Landais Stéphanie , Gounon Pierre , Laurent-Winter Christine , Mazié Jean-Claude , Danchin Antoine , Bârzu Octavian , Sakamoto Hiroshi 
Published: December 2019

ABSTRACT

Mono- and polyclonal antibodies directed against UMP kinase from
Escherichia coli
were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole
E. coli
cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.
PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of
Pseudomonas oleovorans
GPo1 Involved in the Regulatory Expression System for
pha
Genes
Authors: Prieto Maria A. , Bühler Bruno , Jung Kuno , Witholt Bernard , Kessler Birgit 
Published: December 2019

ABSTRACT

The
phaC1
gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of
Pseudomonas oleovorans
GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant
P. oleovorans
strain carrying a
phaC1
::
lacZ
reporter system, that the
phaC1
gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a
P. oleovorans
GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn
5
insertional mutagenesis. Characterization of this mutant allowed us to conclude that
phaF
, a gene located downstream of the
pha
gene cluster, was knocked out in this strain.
P. oleovorans
GPG-Tc6 regained the ability to control
phaC1
gene expression when complemented with the
phaF
wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and
phaI
and
phaF
genes. The amino acid sequences of the
phaI
gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing
phaF
, i.e.,
phaF
and
phaIF
transcripts. Expression of the
phaIF
operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by
P. oleovorans
. A model for the role of PhaF in regulating PHA synthesis is presented.
NolL of
Rhizobium
sp. Strain NGR234 Is Required for
O
-Acetyltransferase Activity
Authors: Berck S. , Perret X. , Quesada-Vincens D. , Promé J.-C. , Broughton W. J. , Jabbouri S. 
Published: December 2019

ABSTRACT

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium
Rhizobium
sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible
nolL
gene, which is preceded by a
nod
box, results in the synthesis of NodNGR factors that lack the 3-
O
- or 4-
O
-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩ
nolL
is not impaired, whereas introduction of the
nod
box::
nolL
construct into the related strain
Rhizobium fredii
USDA257 extends the host range of this bacterium to
Calopogonium caeruleum
,
Leucaena leucocephala
, and
Lotus halophilus
. Nod factors produced by a USDA257(p
nolL
) transconjugant were also acetylated. The
nod
box::
nolL
construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the
nodD1
gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.
Hydride-Meisenheimer Complex Formation and Protonation as Key Reactions of 2,4,6-Trinitrophenol Biodegradation by
Rhodococcus erythropolis
Authors: Rieger Paul-Gerhard , Sinnwell Volker , Preuß Andrea , Francke Wittko , Knackmuss Hans-Joachim 
Published: December 2019

ABSTRACT

Biodegradation of 2,4,6-trinitrophenol (picric acid) by
Rhodococcus erythropolis
HLPM-1 proceeds via initial hydrogenation of the aromatic ring system. Here we present evidence for the formation of a hydride-Meisenheimer complex (anionic ς-complex) of picric acid and its protonated form under physiological conditions. These complexes are key intermediates of denitration and productive microbial degradation of picric acid. For comparative spectroscopic identification of the hydride complex, it was necessary to synthesize this complex for the first time. Spectroscopic data revealed the initial addition of a hydride ion at position 3 of picric acid. This hydride complex readily picks up a proton at position 2, thus forming a reactive species for the elimination of nitrite. Cell extracts of
R. erythropolis
HLPM-1 transform the chemically synthesized hydride complex into 2,4-dinitrophenol. Picric acid is used as the sole carbon, nitrogen, and energy source by
R. erythropolis
HLPM-1.