bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autoregulation of the pTF-FC2 Proteic Poison-Antidote Plasmid Addiction System (
pas
) Is Essential for Plasmid Stabilization
Authors: Smith Anthony S. G. , Rawlings Douglas E. 
Published: December 2019

ABSTRACT

The
pasABC
genes of the proteic plasmid addiction system of broad-host-range plasmid pTF-FC2 were autoregulated. The PasA antidote was able to repress the operon 25-fold on its own, and repression was increased to 100-fold when the PasB toxin was also present. Autoregulation appears to be an essential requirement for
pas
-mediated plasmid stabilization because when the
pas
genes were placed behind the isopropyl-β-
d
-thiogalactopyranoside (IPTG)-regulated
tac
promoter, they were unable to stabilize a heterologous test plasmid.
Int-B13, an Unusual Site-Specific Recombinase of the Bacteriophage P4 Integrase Family, Is Responsible for Chromosomal Insertion of the 105-Kilobase
clc
Element of
Pseudomonas
sp. Strain B13
Authors: Ravatn Roald , Studer Sonja , Zehnder Alexander J. B. , Roelof van der Meer Jan 
Published: December 2019

ABSTRACT

Pseudomonas
sp. strain B13 carries the
clcRABDE
genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb
clc
element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (
glyV
) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (
int-B13
) was located near the right end of the
clc
element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in
Pseudomonas aeruginosa
PaK1. Similar to the bacteriophages φR73 and P4, the
clc
element integrates into the 3′ end of the target tRNA gene. This target site was characterized from four different recipient strains into which the
clc
element integrated, showing sequence specificity of the integration. In
Pseudomonas
sp. strain B13, a circular form of the
clc
element, which carries an 18-bp DNA sequence identical to the 3′-end portion of
glyV
as part of its attachment site (
attP
), could be detected. Upon chromosomal integration of the
clc
element into a bacterial attachment site (
attB
), a functional
glyV
was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient
Escherichia coli
with two recombinant plasmids, one carrying the
int-B13
gene and the
attP
site and the other carrying the
attB
site of
Pseudomonas putida
F1.
Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in
Pseudomonas aeruginosa
Authors: Nishijyo Takayuki , Park Seung-Moon , Lu Chung-Dar , Itoh Yoshifumi , Abdelal Ahmed T. 
Published: December 2019

ABSTRACT

The complete nucleotide sequence for the
aot
operon of
Pseudomonas aeruginosa
PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these,
aotJ
,
aotQ
,
aotM
, and
aotP
, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The
aotO
gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an
aotJ
::
lacZ
translational fusion showed that expression of the
aot
operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible
aru
promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in
P. aeruginosa
. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.
Wound-Released Chemical Signals May Elicit Multiple Responses from an
Agrobacterium tumefaciens
Strain Containing an Octopine-Type Ti Plasmid
Authors: Kalogeraki Virginia S. , Winans Stephen C. 
Published: December 2019

ABSTRACT

The
vir
regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from
Agrobacterium tumefaciens
to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two
vir
regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb
virH
locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The
virH
operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated
virK
. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the
vir
regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to
lacZ
. Expression of these genes was also measured by nuclease S1 protection assays.
virK
and two nonconserved genes, designated
virL
and
virM
, were strongly induced by the
vir
gene inducer acetosyringone. Disruptions of
virH
,
virK
,
virL
, or
virM
did not affect tumorigenesis of
Kalanchöe diagramontiana
leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.
Deletion of the 389 N-Terminal Residues of the Transcriptional Activator AREA Does Not Result in Nitrogen Metabolite Derepression in
Aspergillus nidulans
Authors: Caddick Mark X. , Arst Herbert N. 
Published: December 2019

ABSTRACT

Utilizing a homologous gene replacement in order to retain the native promoter and 5′ and 3′ untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression in
Aspergillus nidulans
. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649–6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.
Transcriptional Regulation of the
Streptococcus salivarius
57.I Urease Operon
Authors: Chen Yi-Ywan M. , Weaver Cheryl A. , Mendelsohn David R. , Burne Robert A. 
Published: December 2019

ABSTRACT

The
Streptococcus salivarius
57.I
ure
cluster was organized as an operon, beginning with
ureI
, followed by
ureABC
(structural genes) and
ureEFGD
(accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A ς
70
-like promoter could be mapped 5′ to
ureI
(
PureI
) by primer extension analysis. The intensity of the signal increased when cells were grown at an acidic pH and was further enhanced by excess carbohydrate. To determine the function(s) of two inverted repeats located 5′ to
PureI
, transcriptional fusions of the full-length promoter region (
PureI
), or a deletion derivative (
PureI
Δ100), and a promoterless chloramphenicol acetyltransferase (CAT) gene were constructed and integrated into the chromosome to generate strains
PureI
CAT and
PureI
Δ100CAT, respectively. CAT specific activities of
PureI
CAT were repressed at pH 7.0 and induced at pH 5.5 and by excess carbohydrate. In
PureI
Δ100CAT, CAT activity was 60-fold higher than in
PureI
CAT at pH 7.0 and pH induction was nearly eliminated, indicating that expression was negatively regulated. Thus, it was concluded that
PureI
was the predominant, regulated promoter and that regulation was governed by a mechanism differing markedly from other known mechanisms for bacterial urease expression.
trans
-Complementation of a
Staphylococcus aureus agr
Mutant by
Staphylococcus lugdunensis agr
RNAIII
Authors: Benito Yvonne , Lina Gerard , Greenland Timothy , Etienne Jerome , Vandenesch François 
Published: December 2019

ABSTRACT

RNAIII from
Staphylococcus lugdunensis
(RNAIII-sl) in a
Staphylococcus aureus agr
mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5′ end of RNAIII-sl and the 3′ end of RNAIII from
S. aureus
restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.
Molecular Characterization of the
Lactococcus lactis Lla
KR2I Restriction-Modification System and Effect of an IS
982
Element Positioned between the Restriction and Modification Genes
Authors: Twomey Denis P. , McKay Larry L. , O’Sullivan Daniel J. 
Published: December 2019

ABSTRACT

The nucleotide sequence of the plasmid-encoded
Lla
KR2I restriction-modification (R-M) system of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (
llaKR2IR
) and methyltransferase (
llaKR2IM
) genes; located in the intergenic region is a copy of the insertion element IS
982
, whose putative transposase gene is codirectionally transcribed with
llaKR2IM
. The deduced sequence of the
Lla
KR2I endonuclease shared homology with the type II endonuclease
Sau
3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5′ GATC 3′. In addition, M ·
Lla
KR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M ·
Sau
3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M ·
Lla
KR2I, all remained undigested after treatment with
Sau
3AI despite the presence of multiple 5′ GATC 3′ sites. The combination of these data suggested that the specificity of the
Lla
KR2I R-M system was likely to be 5′ GATC 3′, with the cytosine residue being modified to 5-methylcytosine. The IS
982
element located within the
Lla
KR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the
llaKR2IR
gene, was shown to overlap the direct repeat sequence on the other side of IS
982
. Specific deletion of IS
982
and one of these direct repeats via a PCR strategy indicated that the
Lla
KR2I R-M determinants do not rely on elements within IS
982
for expression and that the efficiency of bacteriophage restriction was not impaired.
Transcriptional Activation of the
Bacillus subtilis ackA
Gene Requires Sequences Upstream of the Promoter
Authors: Turinsky Andrew J. , Grundy Frank J. , Kim Jeong-Ho , Chambliss Glenn H. , Henkin Tina M. 
Published: December 2019

ABSTRACT

Transcriptional activation of the
Bacillus subtilis ackA
gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the
ackA
promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in
B. subtilis
and other gram-positive bacteria, recognizes a
cis
-acting consensus sequence, designated
cre
(catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions −116.5 and −56.5 of the
ackA
promoter and have been termed
cre1
and
cre2
, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study,
cre2
, the site closer to the promoter, and the region upstream of
cre2
were shown to be indispensable for CcpA-dependent transcriptional activation of
ackA
, whereas
cre1
was not required. In addition, insertion of 5 bp between
cre2
and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of
cre2
and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro to
cre2
but not
cre1
, consistent with the genetic data. Activation of
ackA
transcription was blocked in a
ptsH1/crh
double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.
The Carboxyl Terminus of Protein TraD Adds Specificity and Efficiency to F-Plasmid Conjugative Transfer
Authors: Sastre J. Ignacio , Cabezón Elena , de la Cruz Fernando 
Published: December 2019

ABSTRACT

We isolated and characterized
traD
mutants with an altered specificity of interaction with relaxosomes of various conjugative (F and R388) and mobilizable (RSF1010 and ColE1) plasmids. The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer.
Cyanobacterial Transposons Tn
5469
and Tn
5541
Represent a Novel Noncomposite Transposon Family
Authors: Schaefer Michael R. , Kahn Katherine 
Published: December 2019

ABSTRACT

A noncomposite transposon, designated Tn
5541
, was isolated from strain Fd33 of the filamentous cyanobacterium
Fremyella diplosiphon
UTEX 481. Sequence analysis showed that Tn
5541
is structurally and genetically very similar to Tn
5469
, which is also endogenous to
F. diplosiphon
. Both Tn
5469
and Tn
5541
encode homologous forms of an unusual composite transposase and a protein of unknown function. DNA hybridization analysis showed that like Tn
5469
, Tn
5541
was not widely distributed among cyanobacterial genera. A similar analysis showed that Tn
5469
and Tn
5541
were equally limited to and present as multiple genomic copies in three of six distinct strains comprising the
Tolypothrix
1 cluster of heterocyst-forming filamentous cyanobacteria. These and other distinguishing features suggest that Tn
5469
and Tn
5541
represent a novel noncomposite transposon family.
Campylobacter fetus
Surface Layer Proteins Are Transported by a Type I Secretion System
Authors: Thompson Stuart A. , Shedd Omer L. , Ray Kevin C. , Beins Michael H. , Jorgensen Jesse P. , Blaser Martin J. 
Published: December 2019

ABSTRACT

The virulence of
Campylobacter fetus
, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B
C. fetus
lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by
sapA
homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two
sapA
homologs. Cloning and sequencing of the entire 6.2-kb invertible region from
C. fetus
23D revealed a probable 5.6-kb operon of four overlapping genes (
sapCDEF
, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from
sapA
. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by
Escherichia coli
HlyBD/TolC or
Erwinia chrysanthemi
PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four
C. fetus
SLPs revealed conserved structures that are potential secretion signals. A
C. fetus sapD
mutant neither produced nor secreted SLPs.
E. coli
expressing
C. fetus sapA
and
sapCDEF
secreted SapA, indicating that the
sapCDEF
genes are sufficient for SLP secretion.
C. fetus
SLPs therefore are transported to the cell surface by a type I secretion system.
The EII
Glc
Protein Is Involved in Glucose-Mediated Activation of
Escherichia coli gapA
and
gapB-pgk
Transcription
Authors: Charpentier B. , Bardey V. , Robas N. , Branlant C. 
Published: December 2019

ABSTRACT

The
Escherichia coli gapB
gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH). In most bacteria, the gene for GAPDH is located upstream of the
pgk
gene encoding 3-phosphoglycerate kinase (PGK). This is the case for
gapB
. However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH). The active GAPDH is encoded by the
gapA
gene. Since we found that the nucleotide region upstream of the
gapB
open reading frame is responsible for part of the PGK production, we analyzed
gapB
promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription. We showed the presence of a unique transcription promoter,
gapB
P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site. Interestingly, the
gapB
P0 promoter activity was strongly enhanced when glucose was used as the carbon source. In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter
gapB
P0 activity. In contrast, abolition of CRP production or of cAMP biosynthesis (
crp
or
cya
mutant strains) strongly reduced promoter
gapB
P0 activity. This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter
gapB
P0 activity. We also showed that glucose stimulation of
gapB
P0 promoter activity depends on the expression of enzyme II
Glc
(EII
Glc
), encoded by the
ptsG
gene, and that the
gapA
P1 promoter is also activated by glucose via the EII
Glc
protein. A similar glucose-mediated activation, dependent on the EII
Glc
protein, was described by others for the
pts
operon. Altogether, this shows that when glucose is present in the growth medium expression of the
E. coli
genes required for its uptake (
pts
) and its metabolism (
gapA
and
gapB-pgk
) are coordinately activated by a mechanism dependent upon the EII
Glc
protein.
Region of Flo1 Proteins Responsible for Sugar Recognition
Authors: Kobayashi Osamu , Hayashi Nobuyuki , Kuroki Ryota , Sone Hidetaka 
Published: December 2019

ABSTRACT

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The
FLO1
gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of
FLO1
, named Lg-
FLO1
, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both
FLO1
and Lg-
FLO1
are involved in the yeast flocculation phenomenon, the
FLO1
gene in the mannose-specific flocculation strain was replaced by the Lg-
FLO1
gene. The transformant in which the Lg-
FLO1
gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the
FLO1
and Lg-
FLO1
genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric
FLO1
and Lg-
FLO1
genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.
A MADS Box Protein Consensus Binding Site Is Necessary and Sufficient for Activation of the Opaque-Phase-Specific Gene
OP4
of
Candida albicans
Authors: Lockhart Shawn R. , Nguyen Mau , Srikantha Thyagarajan , Soll David R. 
Published: December 2019

ABSTRACT

The majority of strains of
Candida albicans
can switch frequently and reversibly between two or more general phenotypes, a process now considered a putative virulence factor in this species.
Candida albicans
WO-1 switches frequently and reversibly between a white and an opaque phase, and this phenotypic transition is accompanied by the differential expression of white-phase-specific and opaque-phase-specific genes. In the opaque phase, cells differentially express the gene
OP4
, which encodes a putative protein 402 amino acids in length that contains a highly hydrophobic amino-terminal sequence and a carboxy-terminal sequence with a pI of 10.73. A series of deletion constructs fused to the
Renilla reniformis
luciferase was used to functionally characterize the
OP4
promoter in order to investigate how this gene is differentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between −422 and −404 bp. This sequence contained a MADS box consensus binding site, most closely related to the Mcm1 binding site of
Saccharomyces cerevisiae
. A number of point mutations generated in the MADS box consensus binding site as well as a complete deletion of the consensus site further demonstrated that it was essential for the activation of
OP4
transcription in the opaque phase. Gel mobility shift assays with the 17-bp activation sequence identified three specific complexes which formed with both white- and opaque-phase cell extracts. Competition with a putative MADS box consensus binding site from the promoter of the coordinately regulated opaque-phase-specific gene
PEP1 (SAP1)
and the human MADS box consensus binding site for serum response factor demonstrated that one of the three complexes formed was specific to the
OP4
sequence.