bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism inLactobacillus pentosus
Authors: Chaillou Stéphane , Lokman B. Christien , Leer Rob J. , Posthuma Clara , Postma Pieter W. , Pouwels Peter H. 
Published: December 2019

ABSTRACTTwo genes,xylPandxylQ, from the xylose regulon ofLactobacillus pentosuswere cloned and sequenced. Together with the repressor gene of the regulon,xylR, thexylPQgenes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream ofxylP. A putativexylRbinding site (xylO) and acre-like element, mediating CcpA-dependent catabolite repression, were found in the promoter region.L. pentosusmutants in which bothxylPandxylQ(LPE1) or onlyxylQ(LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (α-d-xylopyranosyl-(1,6)-d-glucopyranose). Disruption ofxylQresulted specifically in the loss of a membrane-associated α-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria, α-xylosidase could catalyze the hydrolysis of isoprimeverose andp-nitrophenyl-α-d-xylopyranoside with apparentKmandVmaxvalues of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the α-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-α-d-xylopyranoside nor α-glucosides were substrates. Glucose repressed the synthesis of α-xylosidase fivefold, and 80% of this repression was released in anL. pentosus ΔccpAmutant. The α-xylosidase gene was also expressed in the absence of xylose whenxylRwas disrupted.
Structure and Expression of the FlaA Periplasmic Flagellar Protein ofBorrelia burgdorferi
Authors: Ge Yigong , Li Chunhao , Corum Linda , Slaughter Clive A. , Charon Nyles W. 
Published: December 2019

ABSTRACTThe spirochete which causes Lyme disease,Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of theB. burgdorferiflagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that aflaAhomolog was expressed inB. burgdorferiand that it mapped in afla/cheoperon. These results led us to analyze the PFs and FlaA ofB. burgdorferiin detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purifyB. burgdorferiPFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes,B. burgdorferiFlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart fromSerpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant ofB. burgdorferi(HB19Fla−). Although this mutant still synthesizedflaAmessage in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis usingTreponema pallidumanti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs ofB. burgdorferiand that they are regulated differently from flagellin proteins of other bacteria.
Isolation of an
Escherichia coli
K-12 Mutant Strain Able To Form Biofilms on Inert Surfaces: Involvement of a New
ompR
Allele That Increases Curli Expression
Authors: Vidal Olivier , Longin Robert , Prigent-Combaret Claire , Dorel Corinne , Hooreman Michel , Lejeune Philippe 
Published: December 2019

ABSTRACT

Classical laboratory strains of
Escherichia coli
do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized
E. coli
K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652–655, 1989). The production of curli (visualized by Congo red binding) and the expression of the
csgA
gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the
ompR
allele described in this work. Transduction of knockout mutations in either
csgA
or
ompR
caused the loss of the adherence properties of several biofilm-forming
E. coli
strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.
Regulation of Benzoate Degradation in
Acinetobacter
sp. Strain ADP1 by BenM, a LysR-Type Transcriptional Activator
Authors: Collier Lauren S. , Gaines George L. , Neidle Ellen L. 
Published: December 2019

ABSTRACT

In
Acinetobacter
sp. strain ADP1, benzoate degradation requires the
ben
genes for converting benzoate to catechol and the
cat
genes for degrading catechol. Here we describe a novel transcriptional activator, BenM, that regulates the chromosomal
ben
and
cat
genes. BenM is homologous to CatM, a LysR-type transcriptional activator of the
cat
genes. Unusual regulatory features of this system include the abilities of both BenM and CatM to recognize the same inducer,
cis
,
cis
-muconate, and to regulate some of the same genes, such as
catA
and
catB
. Unlike CatM, BenM responded to benzoate. Benzoate together with
cis
,
cis
-muconate increased the BenM-dependent expression of the
benABCDE
operon synergistically. CatM was not required for this synergism, nor did CatM regulate the expression of a chromosomal
benA
::
lacZ
transcriptional fusion. BenM-mediated regulation differs significantly from that of the TOL plasmid-encoded conversion of benzoate to catechol in pseudomonads. The
benM
gene is immediately upstream of, and divergently transcribed from,
benA
, and a possible DNA binding site for BenM was identified between the two coding regions. Two mutations in the predicted operator/promoter region rendered
ben
gene expression either constitutive or inducible by
cis
,
cis
-muconate but not benzoate. Mutants lacking BenM, CatM, or both of these regulators degraded aromatic compounds at different rates, and the levels of intermediary metabolites that accumulated depended on the genetic background. These studies indicated that BenM is necessary for
ben
gene expression but not for expression of the
cat
genes, which can be regulated by CatM. In a
catM
-disrupted strain, BenM was able to induce higher levels of
catA
expression than
catB
expression.
A Seven-Gene Locus for Synthesis of Phenazine-1-Carboxylic Acid by
Pseudomonas fluorescens
2-79
Authors: Mavrodi Dmitri V. , Ksenzenko Vladimir N. , Bonsall Robert F. , Cook R. James , Boronin Alexander M. , Thomashow Linda S. 
Published: December 2019

ABSTRACT

Pseudomonas fluorescens
2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated
phzABCDEFG
that are sufficient for synthesis of PCA were localized within a 6.8-kb
Bgl
II-
Xba
I fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all
phz
genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the
phzC
,
phzD
, and
phzE
genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5′-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the
phzA
and
phzB
genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes,
phzX
and
phzY
, that are homologous to
phzA
and
phzB
, respectively, were cloned and sequenced from
P. aureofaciens
30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the
phz
genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.
3-Nitroadipate, a Metabolic Intermediate for Mineralization of 2,4-Dinitrophenol by a New Strain of a
Rhodococcus
Species
Authors: Blasco Rafael , Moore Edward , Wray Victor , Pieper Dietmar , Timmis Kenneth , Castillo Francisco 
Published: December 2019

ABSTRACT

The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a
Rhodococcus
species closely related to
Rhodococcus opacus. Rhodococcus
sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by
1
H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.
Function and Regulation of
glnA
in the Methanogenic Archaeon
Methanococcus maripaludis
Authors: Cohen-Kupiec Rachel , Marx Christopher J. , Leigh John A. 
Published: December 2019

ABSTRACT

The
glnA
gene in the domains
Bacteria
and
Archaea
encodes glutamine synthetase, a universally distributed enzyme that functions in ammonia assimilation and glutamine synthesis. We investigated the regulation and function of
glnA
in the methanogenic archaeon
Methanococcus maripaludis
. The deduced amino acid sequence of the gene demonstrated its membership in class GSI-α of glutamine synthetases. The gene appeared to be expressed as a monocistronic operon.
glnA
mRNA levels and specific activities of glutamine synthetase were regulated similarly by nitrogen. Three transcription start sites were identified, corresponding to two overlapping nitrogen-regulated promoters and one weaker constitutive promoter. An inverted repeat immediately upstream of the regulated transcription start sites mediated repression under noninducing conditions. Thus, mutations that altered the sequence of the inverted repeat resulted in derepression. The inverted repeat had sequence similarity with a repeat that we previously identified as the
nif
operator of
M. maripaludis
, suggesting a common mechanism of nitrogen regulation. Efforts to produce a
glnA
null mutant failed, suggesting that
glnA
is an essential gene in
M. maripaludis
.
Peptidoglycan Fine Structure of the Radiotolerant Bacterium
Deinococcus radiodurans
Sark
Authors: Quintela José Carlos , García-del Portillo Francisco , Pittenauer Ernst , Allmaier Günter , de Pedro Miguel A. 
Published: December 2019

ABSTRACT

Peptidoglycan from
Deinococcus radiodurans
was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was:
N
-acetylglucosamine–
N
-acetylmuramic acid–
l
-Ala–
d
-Glu-(γ)–
l
-Orn-[(δ)Gly-Gly]–
d
-Ala–
d
-Ala. Cross-linkage was mediated by (Gly)
2
bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close
Thermus thermophilus
are discussed.
Roles of Chemosensory Pathways in Transient Changes in Swimming Speed of
Rhodobacter sphaeroides
Induced by Changes in Photosynthetic Electron Transport
Authors: Romagnoli Simona , Armitage Judith P. 
Published: December 2019

ABSTRACT

The response of free-swimming
Rhodobacter sphaeroides
to increases and decreases in the intensity of light of different wavelengths was analyzed. There was a transient (1 to 2 s) increase in swimming speed in response to an increase in light intensity, and there was a similar transient stop when the light intensity decreased. Measurement of changes in membrane potential and the use of electron transport inhibitors showed that the transient increase in swimming speed, following an increase in light intensity, and the stop following its decrease were the result of changes in photosynthetic electron transport.
R. sphaeroides
has two operons coding for multiple homologs of the enteric chemosensory genes. Mutants in the first chemosensory operon showed wild-type photoresponses. Mutants with the
cheA
gene of the second operon (
cheA
II
) deleted, either with or without the first operon present, showed inverted photoresponses, with free-swimming cells stopping on an increase in light intensity and increasing swimming speed on a decrease. These mutants also lacked adaptation. Transposon mutants with mutations in
cheA
II
, which also reduced expression of downstream genes, however, showed no photoresponses. These results show that (i) free-swimming cells respond to both an increase and a decrease in light intensity (tethered cells only show the stopping on a step down in light intensity), (ii) the signal comes from photosynthetic electron transfer, and (iii) the signal is primarily channelled through the second chemosensory pathway. The different responses shown by the
cheA
II
deletion and insertion mutants suggest that CheW
II
is required for photoresponses, and a third sensory pathway can substitute for CheA
II
as long as CheW
II
is present. The inverted response suggests that transducers are involved in photoresponses as well as chemotactic responses.
Cloning and Expression of Three New
Azotobacter vinelandii
Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases
Authors: Svanem Britt Iren Glærum , Skjåk-Bræk Gudmund , Ertesvåg Helga , Valla Svein 
Published: December 2019

ABSTRACT

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from
Azotobacter vinelandii
(
algE1
to -
5
) has previously been reported. The corresponding proteins catalyze the Ca
2+
-dependent polymer-level epimerization of β-
d
-mannuronic acid to α-
l
-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated
algE6
,
algE7
, and
algY
. All three genes were sequenced and expressed in
Escherichia coli
. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in
algE4
expressed an active epimerase. Southern blot analysis of genomic
A. vinelandii
DNA, using the 5′ part of
algE2
as a probe, indicated that all hybridization signals originated from
algE1
to -
5
or the three new genes reported here.
Phosphate Control of Oxytetracycline Production by
Streptomyces rimosus
Is at the Level of Transcription from Promoters Overlapped by Tandem Repeats Similar to Those of the DNA-Binding Sites of the OmpR Family
Authors: McDowall Kenneth J. , Thamchaipenet Arinthip , Hunter Iain S. 
Published: December 2019

ABSTRACT

Physiological studies have shown that
Streptomyces rimosus
produces the polyketide antibiotic oxytetracycline abundantly when its mycelial growth is limited by phosphate starvation. We show here that transcripts originating from the promoter for one of the biosynthetic genes,
otcC
(encoding anhydrotetracycline oxygenase), and from a promoter for the divergent
otcX
genes peak in abundance at the onset of antibiotic production induced by phosphate starvation, indicating that the synthesis of oxytetracycline is controlled, at least in part, at the level of transcription. Furthermore, analysis of the sequences of the promoters for
otcC
,
otcX
, and the polyketide synthase (
otcY
) genes revealed tandem repeats having significant similarity to the DNA-binding sites of ActII-Orf4 and DnrI, which are
Streptomyces
antibiotic regulatory proteins (SARPs) related to the OmpR family of transcription activators. Together, the above results suggest that oxytetracycline production by
S. rimosus
requires a SARP-like transcription factor that is either produced or activated or both under conditions of low phosphate concentrations. We also provide evidence consistent with the
otrA
resistance gene being cotranscribed with
otcC
as part of a polycistronic message, suggesting a simple mechanism of coordinate regulation which ensures that resistance to the antibiotic increases in proportion to production.
MHY1
Encodes a C
2
H
2
-Type Zinc Finger Protein That Promotes Dimorphic Transition in the Yeast
Yarrowia lipolytica
Authors: Hurtado Cleofe A. R. , Rachubinski Richard A. 
Published: December 2019

ABSTRACT

The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi. Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions. We have isolated and characterized a gene,
MHY1
, whose transcription is dramatically increased during the yeast-to-hypha transition in
Yarrowia lipolytica
. Deletion of
MHY1
is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth.
MHY1
encodes a C
2
H
2
-type zinc finger protein, Mhy1p, which can bind putative
cis
-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor. Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip.
Escherichia coli
DNA Topoisomerase I Copurifies with Tn
5
Transposase, and Tn
5
Transposase Inhibits Topoisomerase I
Authors: Yigit Hesna , Reznikoff William S. 
Published: December 2019

ABSTRACT

Tn
5
transposase (Tnp) overproduction is lethal to
Escherichia coli
. Genetic evidence suggested that this killing involves titration of
E. coli
topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn
5
Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Δ37Tnp and Δ55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Δ37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a
topA
mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp.
Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from
Bacillus thuringiensis
Authors: Wilcks Andrea , Smidt Lasse , Økstad Ole Andreas , Kolstø Anne-Brit , Mahillon Jacques , Andrup Lars 
Published: December 2019

ABSTRACT

A 5.8-kb fragment of the large conjugative plasmid pAW63 from
Bacillus thuringiensis
subsp.
kurstaki
HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63
ori
. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the
Enterococcus faecalis
plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes
repC
and
prgO
, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in
Bacillus subtilis
and
B. thuringiensis
and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other
B. thuringiensis
subsp.
kurstaki
strains and to a strain of
B. thuringiensis
subsp.
alesti
.
In Vitro Selection of Integration Host Factor Binding Sites
Authors: Goodman Steven D. , Velten Nerissa J. , Gao Qian , Robinson Scott , Segall Anca M. 
Published: December 2019

ABSTRACT
Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3′ domain is conserved, the 5′ domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5′ domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5′ binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5′ binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.