bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Cold-Box Region in the 5′ Untranslated Region of the
cspA
mRNA in Its Transient Expression at Low Temperature in
Escherichia coli
Authors: Fang Li , Hou Yan , Inouye Masayori 
Published: December 2019

ABSTRACT

Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced in
Escherichia coli
. However, when the 5′ untranslated region (5′ UTR) of
cspA
mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the “cold box” existing in the 5′ UTRs of
cspA
,
cspB
, and
csdA
. Here, we demonstrate that the overproduction of the 5′ UTR of not only
cspA
but also
cspB
and
csdA
mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the
cspA
5′ UTR its derepression function was abolished. The amount of mRNA from the chromosomal
cspA
gene was much higher in cells overproducing the wild-type 5′ UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5′ UTR. The stability of the chromosomal
cspA
mRNA in cells overproducing the wild-type 5′ UTR was almost identical to that in cells overproducing the cold-box-deleted 5′ UTR. Therefore, the derepression of
cspA
caused by overproduction of 5′ UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in
cspA
transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with a
cspA
mutant strain containing a cold-box-deleted
cspA
gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.
Roles of DctA and DctB in Signal Detection by the Dicarboxylic Acid Transport System of
Rhizobium leguminosarum
Authors: Reid Colm J. , Poole Philip S. 
Published: December 2019

ABSTRACT

The
dctA
gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for
dctB
and
dctD
. The
dctA
promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader.
dctAp
also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in the
dctA-dctB
intergenic region (
dctA101
) which locks transcription of
dctA
at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix
−
) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation of
dctB
or
dctD
in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.
Unconventional Genomic Organization in the Alpha Subgroup of the
Proteobacteria
Authors: Jumas-Bilak Estelle , Michaux-Charachon Sylvie , Bourg Gisele , Ramuz Michel , Allardet-Servent Annick 
Published: December 2019

ABSTRACT

Pulsed-field gel electrophoresis was used to analyze the genomic organization of 16 bacteria belonging or related to the family
Rhizobiaceae
of the alpha subgroup of the class
Proteobacteria
. The number and sizes of replicons were determined by separating nondigested DNA. Hybridization of an
rrn
gene probe was used to distinguish between chromosomes and plasmids. Members of the genus
Agrobacterium
all possess two chromosomes, and each biovar has a specific genome size. As previously demonstrated for
Agrobacterium tumefaciens
C58, the smaller chromosomes of
Agrobacterium
biovar 1 and
Agrobacterium rubi
strains appear to be linear. The genomes of
Rhizobium
strains were all of similar sizes but were seen to contain either one, two, or three megareplicons. Only one chromosome was present in the member of the related genus
Phyllobacterium
. We found one or two chromosomes in
Rhodobacter
and
Brucella
species, two chromosomes in
Ochrobactrum anthropi
, and one chromosome in
Mycoplana dimorpha
and
Bartonella quintana
; all of these genera are related to the
Rhizobiaceae
. The presence of multiple chromosomes is discussed from a phylogenetic and taxonomic point of view.
Suppressor Scanning at Positions 177 and 236 in the
Escherichia coli
Lactose/H
+
Cotransporter and Stereotypical Effects of Acidic Substituents That Suggest a Favored Orientation of Transmembrane Segments Relative to the Lipid Bilayer
Authors: King Steven C. , Li Suzhen 
Published: December 2019

ABSTRACT
Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents. Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.
Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMP
Rtr
CFN299pc60) in
Rhizobium tropici
Authors: Mavingui Patrick , Laeremans Toon , Flores Margarita , Romero David , Martínez-Romero Esperanza , Palacios Rafael 
Published: December 2019

ABSTRACT

Amplifiable DNA regions (amplicons) have been identified in the genome of
Rhizobium etli
. Here we report the isolation and molecular characterization of a symbiotic amplicon of
Rhizobium tropici
. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of
R. tropici
CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into
Escherichia coli
and then into
Agrobacterium tumefaciens
. The 60-kb symbiotic amplicon, which we termed AMP
Rtr
CFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence IS
Rtr1
. Amplification of AMP
Rtr
CFN299pc60 through homologous recombination between IS
Rtr1
repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless
A. tumefaciens
strain confers on the transconjugant the ability to produce
R. tropici
Nod factors and to nodulate
Phaseolus vulgaris
, indicating that
R. tropici
genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.
Posttranscriptional Modifications in 16S and 23S rRNAs of the Archaeal Hyperthermophile
Sulfolobus solfataricus
Authors: Noon Kathleen R. , Bruenger Eveline , McCloskey James A. 
Published: December 2019

ABSTRACT

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile
Sulfolobus solfactaricus
by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75°C, compared with 11 and 23 sites, respectively, in
Escherichia coli
. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2′ of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83°C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23°C range shown for 2′-
O
-methyladenosine in 16S rRNA (21% increase) and for 2′-
O
-methylcytidine (24%) and 2′-
O
-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.
Evidence for a Conjugation-Like Mechanism of DNA Transfer in
Helicobacter pylori
Authors: Kuipers Ernst J. , Israel Dawn A. , Kusters Johannes G. , Blaser Martin J. 
Published: December 2019

ABSTRACT

Many strains of
Helicobacter pylori
are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among
H. pylori
strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with
H. pylori
cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between
H. pylori
cells.
DNA Synthesis and Viability of a
mutT
Derivative of
Escherichia coli
WP2 under Conditions of Amino Acid Starvation and Relation to Stationary-Phase (Adaptive) Mutation
Authors: Bridges Bryn A. , Ereira Sharon 
Published: December 2019

ABSTRACT

Escherichia coli
WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10
8
on plates lacking the required amino acid. They lose viability for some days, and then a subpopulation recovers and there is cell turnover. At very low plating densities (around 10
2
per plate), almost every cell will eventually form a small but visible colony. At intermediate plating densities (10
6
to 10
7
per plate), there is an immediate increase in the number of viable bacteria. The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species. Mutations in
mutT
bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10
7
per plate). We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10
8
scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid. The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a
polA
derivative. We cannot exclude the possibility that this residual DNA synthesis is peculiar to
mutT
bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain. If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10
7
per plate) and reduced the rate of appearance of mutants at plating densities above 10
8
per plate.
glkA
Is Involved in Glucose Repression of Chitinase Production in
Streptomyces lividans
Authors: Saito Akihiro , Fujii Takeshi , Yoneyama Tadakatsu , Miyashita Kiyotaka 
Published: December 2019

ABSTRACT

Chitinase production in
Streptomyces lividans
is induced by chitin and repressed in the presence of glucose. A mutant of
S. lividans
TK24, strain G015, which was defective in glucose repression of chitinase production, was obtained by screening colonies for zones of clearing on colloidal chitin agar plates containing 1.0% (wt/vol) glucose. The transcriptional analysis of
chiA
in G015 with
xylE
, which encodes catechol 2,3-dioxygenase, as a reporter gene showed that the transcription from the
chiA
promoter of
S. lividans
TK24 occurred regardless of the presence of glucose. G015 was resistant to 2-deoxyglucose (2-DOG) and did not utilize glucose as a sole carbon source. When a DNA fragment containing
glkA
, a gene for glucose kinase, of
Streptomyces coelicolor
A3(2) was introduced into strain G015 on a low-copy-number plasmid, the sensitivity to 2-DOG, the ability to utilize glucose, and the glucose repression of chitinase production were restored. These results indicate that
glkA
is involved in glucose repression of chitinase production in
S. lividans
TK24.
Translation of the Flagellar Gene
fliO
of
Salmonella typhimurium
from Putative Tandem Starts
Authors: Schoenhals Gary J. , Kihara May , Macnab Robert M. 
Published: December 2019

ABSTRACT

The flagellar gene
fliO
of
Salmonella typhimurium
can be translated from an AUG codon that overlaps the termination codon of
fliN
(K. Ohnishi et al., J. Bacteriol. 179:6092–6099, 1997). However, it had been concluded on the basis of complementation analysis that in
Escherichia coli
a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189–197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene
cheA
; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with
cheA
. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in
S. typhimurium
there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A
fliO
null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between
S. typhimurium
and
E. coli
and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.
Mutational Analysis of the TnrA-Binding Sites in the
Bacillus subtilis nrgAB
and
gabP
Promoter Regions
Authors: Wray Lewis V. , Zalieckas Jill M. , Ferson Amy E. , Fisher Susan H. 
Published: December 2019

ABSTRACT

Transcription of the
Bacillus subtilis nrgAB
promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN
7
TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated
nrgAB
,
gabP
P2, and
nas
promoters. Oligonucleotide-directed mutagenesis of the
nrgAB
promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of
nrgAB
expression. Mutations that alter the relative distance between the two half-sites of the
nrgAB
TnrA site abolish nitrogen regulation of
nrgAB
expression. Spacer mutations that change the relative distance between the TnrA site and −35 region of the
nrgAB
promoter reveal that activation of
nrgAB
expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the
gabP
P2 TnrA site showed that this sequence is also required for nitrogen-regulated
gabP
P2 expression. The TnrA protein, expressed in an overproducing
Escherichia coli
strain, had a 625-fold-higher affinity for the wild-type
nrgAB
promoter DNA than for a mutated
nrgAB
promoter DNA fragment that is unable to activate
nrgAB
expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate
nrgAB
expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.
Replicase, Excisionase, and Integrase Genes of the
Streptomyces
Element pSAM2 Constitute an Operon Positively Regulated by the
pra
Gene
Authors: Sezonov Guennadi , Duchêne Anne-Marie , Friedmann Annick , Guérineau Michel , Pernodet Jean-Luc 
Published: December 2019

ABSTRACT

pSAM2 is a site-specific integrative element from
Streptomyces ambofaciens
. The
pra
gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the
pra
gene was placed under the control of the inducible
tipAp
promoter. Transformation of
Streptomyces lividans
by these derivatives was efficient only when
pra
expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the
pra
expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes
repSA
,
xis
, and
int
coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by
pra.
Promoter Region of the
Escherichia coli
O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence
Authors: Marolda Cristina L. , Valvano Miguel A. 
Published: December 2019

ABSTRACT

We report the identification of the promoter region of the
Escherichia coli
O7-specific lipopolysaccharide (LPS) gene cluster (
wb
EcO7
). Typical −10 and −35 sequences were found to be located in the intervening region between
galF
and
rlmB
, the first gene of the
wb
EcO7
cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two
ops
sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless
lac
operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the
ops
sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the
waaQGPSBIJYZK
genes. We observed another predicted stem-loop, located immediately downstream from the
wb
EcO7
transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes.
wb
EcO7
-
lac
transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg
2+
concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the
wb
EcO7
promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.
Folding-Based Suppression of Extracytoplasmic Toxicity Conferred by Processing-Defective LamB
Authors: Cosma Christine L. , Crotwell Michelle D. , Burrows Stephanie Y. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of
Escherichia coli
. Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase. As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane. These mutant porins are toxic when secreted to the cell envelope. Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J. H. Carlson and T. J. Silhavy, J. Bacteriol. 175:3327–3334, 1993). We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism. Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane. Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA. The suppression mediated by TetA′ is independent of the CpxA/CpxR regulon and the ς
E
regulon, both of which are involved in regulating protein trafficking functions in the cell envelope.