bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mycobacterium tuberculosis rrn
Promoters: Differential Usage and Growth Rate-Dependent Control
Authors: Verma Anita , Sampla Avinash K. , Tyagi Jaya Sivaswami 
Published: December 2019

ABSTRACT

Mycobacterium tuberculosis
is a slow-growing pathogen and is characterized by a low content of RNA per unit of DNA. rRNAs represent a major proportion of the total RNA pool, and the entire requirement for rRNA is met by transcription from a single
rrn
operon that is driven by two promoters, P1 and P3. This study attempted to analyze the specific role of the
rrn
promoter in determining the characteristically low levels of RNA in
M. tuberculosis
. For this purpose, the activity of the
M. tuberculosis rrn
promoter as a function of the growth rate was studied by
rrn-lacZ
promoter fusion, hybridization, and primer extension analysis in
M. smegmatis. rrn
promoter signals were faithfully recognized in
M. smegmatis
cultures harboring the
rrn-lacZ
promoter construct. In
M. smegmatis
cultures that displayed doubling times varying between 3.06 and 6.5 h, β-galactosidase activity increased ∼sixfold in proportion to the growth rate (μ). There was a corresponding increase in the amount of
lacZ
-specific mRNA, while the plasmid copy number remained essentially unchanged. For any given μ, the P3 promoter was ∼twofold more efficiently utilized than the P1 promoter. Since both promoters of the
M. tuberculosis rrn
operon are regulatable as a function of growth rate in
M. smegmatis
cultures, it is implied that the inherent structure or sequence of the
rrn
promoter per se is not primarily responsible for the observed lack of modulation of RNA synthesis in
M. tuberculosis.
Mutational Analysis of the
cbb
Operon (CO
2
Assimilation) Promoter of
Ralstonia eutropha
Authors: Jeffke Thomas , Gropp Niels-Holger , Kaiser Claudia , Grzeszik Claudia , Kusian Bernhard , Bowien Botho 
Published: December 2019

ABSTRACT

P
L
promoters direct the transcription of the duplicated
cbb
operons from the facultative chemoautotroph
Ralstonia eutropha
H16. The operons encode most enzymes of the Calvin-Benson-Bassham carbon reduction cycle required for CO
2
assimilation. Their transcription depends on the activator protein CbbR. Structure-function relationships in the cloned chromosomal promoter region were analyzed by site-directed mutagenesis. P
L
was altered in its presumed hexameric −35 and/or −10 box or in the spacer region between the boxes to achieve a greater or lesser resemblance to the structure of the ς
70
consensus promoter of
Escherichia coli
. P
L
::
lacZ
transcriptional fusions of various promoter variants were assayed in transconjugant strains of
R. eutropha
as well as in corresponding
cbbR
deletion mutants. Mutations increasing the similarity of the −35 and/or −10 box to the consensus sequence stimulated P
L
activity to various extents, whereas mutations deviating from the consensus decreased the activity. The length of the spacer region also proved to be critical. The conversion of the boxes, either individually or simultaneously, into the consensus sequences resulted in a highly active P
L
. All improved P
L
mutants, however, retained the activation under inducing or derepressing growth conditions, although the full-consensus promoter was nearly constitutive. They were also activated in the
cbbR
mutants. The activity of the overlapping, divergently oriented
cbbR
promoter was less affected by the mutations. The half- and full-consensus P
L
mutants were comparably active in
E. coli
. Two major conclusions were drawn from the results: (i) the location and function of P
L
were verified, and (ii) indirect evidence was obtained for the involvement of another regulator(s), besides CbbR, in the transcriptional control of the
R. eutropha cbb
operons.
Biochemical and Genetic Evidence for Participation of DevR in a Phosphorelay Signal Transduction Pathway Essential for Heterocyst Maturation in
Nostoc punctiforme
ATCC 29133
Authors: Hagen Kari D. , Meeks John C. 
Published: December 2019

ABSTRACT

In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in
Nostoc punctiforme
ATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate
N. punctiforme
mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox
−
phenotype like the original
devR
insertion mutant UCD 311, consistent with a phosphotransferase role for DevR.
Isolation and Properties of the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL
Authors: Money Tracy , Jones Tamera , Dixon Ray , Austin Sara 
Published: December 2019

ABSTRACT

In
Azotobacter vinelandii
, activation of
nif
gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.
Genomic Relatedness of
Chlamydia
Isolates Determined by Amplified Fragment Length Polymorphism Analysis
Authors: Meijer Adam , Morré Servaas A. , Van Den Brule Adriaan J. C. , Savelkoul Paul H. M. , Ossewaarde Jacobus M. 
Published: December 2019

ABSTRACT

The genomic relatedness of 19
Chlamydia pneumoniae
isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21
Chlamydia trachomatis
isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6
Chlamydia psittaci
isolates (5 avian isolates and 1 feline isolate), and 1
Chlamydia pecorum
isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical
C. trachomatis
isolates. The fingerprints of all
C. pneumoniae
isolates were nearly identical, clustering together at a Dice similarity of 92.6% (± 1.6% standard deviation). The fingerprints of the
C. trachomatis
isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The
C. psittaci
fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of
C. pecorum
was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular,
omp1
and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of
C. pneumoniae
is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.
Cloning, Sequence, and Transcriptional Regulation of the Operon Encoding a Putative N -Acetylmannosamine-6-Phosphate Epimerase (
nanE
) and Sialic Acid Lyase (
nanA
) in
Clostridium perfringens
Authors: Walters Dana M. , Stirewalt Veronica L. , Melville Stephen B. 
Published: December 2019

ABSTRACT

Clostridium perfringens
can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and
N
-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts
N
-acetylmannosamine-6-phosphate to
N
-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from
C. perfringens
NCTC 8798 that contains the
nanE
and
nanA
genes has been cloned. The identification of the
nanA
gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a
nanA Escherichia coli
strain, EV78. The
nanA
gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the
nanE
and
nanA
genes comprise an operon and that transcription of the operon in
C. perfringens
is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of
nanE-nanA
transcription by glucose. With a plasmid construct containing a promoterless
cpe-gusA
gene fusion, in which β-glucuronidase activity indicated that the
gusA
gene acted as a reporter for transcription, a promoter was localized to the region upstream of the
nanE
gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the
nanE
coding sequence.
Functional Analysis of the Carbohydrate-Binding Domains of
Erwinia chrysanthemi
Cel5 (Endoglucanase Z) and an
Escherichia coli
Putative Chitinase
Authors: Simpson Helen D. , Barras Frederic 
Published: December 2019

ABSTRACT

The Cel5 cellulase (formerly known as endoglucanase Z) from
Erwinia chrysanthemi
is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBD
Cel5
has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBD
Cel5
exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from
Escherichia coli
. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the
E. coli
chromosome and purified by metal chelating chromatography. While CBD
Cel5
bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.
Effects of Growth Conditions on Expression of Mycobacterial
murA
and
tyrS
Genes and Contributions of Their Transcripts to Precursor rRNA Synthesis
Authors: Gonzalez-y-Merchand J. A. , Colston M. J. , Cox R. A. 
Published: December 2019

ABSTRACT

All mycobacteria studied to date have an rRNA operon, designated
rrnA
, located downstream from a single copy of the
murA
gene, which encodes an enzyme (EC
2.5.1.7
) important for peptidoglycan synthesis. The
rrnA
operon has a promoter, P1(A), located within the coding region of
murA
, near the 3′ end. Samples of RNA were isolated from
Mycobacterium tuberculosis
at different stages of the growth cycle and from
Mycobacterium smegmatis
grown under different conditions. RNase protection assays were used to investigate transcripts of both
murA
and
rrnA
. Transcription of
murA
was found to continue into the 16S rRNA gene, as if
murA
and
rrnA
form a hybrid (protein coding-rRNA coding) operon. During the growth of
M. tuberculosis
, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of
M. smegmatis
RNA revealed that the level of
murA
RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for
murA
and
rrnA
.
M. smegmatis
has a second rRNA operon,
rrnB
, located downstream from a single copy of the
tyrS
gene, encoding tyrosyl-tRNA synthetase. Transcription of
tyrS
was found to continue into the 16S rRNA gene
rrnB
. The hybrid
tyrS-rrnB
operon contributed 0.2 to 0.6% to
rrnB
transcripts. The pattern of
tyrS
expression during the growth cycle matched the pattern of
rrnB
expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis.
Three Distinct Phases of Isoprene Formation during Growth and Sporulation of
Bacillus subtilis
Authors: Wagner William P. , Nemecek-Marshall Michele , Fall Ray 
Published: December 2019

ABSTRACT

During growth on a glucose-tryptone medium,
Bacillus subtilis
6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1,3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.
An Unspliced Group I Intron in 23S rRNA LinksChlamydiales, Chloroplasts, and Mitochondria
Authors: Everett Karin D. E. , Kahane Simona , Bush Robin M. , Friedman Maureen G. 
Published: December 2019

ABSTRACT

Chlamydia was the only genus in the orderChlamydiales until the recent characterization ofSimkania negevensis ZT and Parachlamydia acanthamoebae strains. The present study ofChlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU · 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.
The icfG Gene Cluster ofSynechocystis sp. Strain PCC 6803 Encodes an Rsb/Spo-Like Protein Kinase, Protein Phosphatase, and Two Phosphoproteins
Authors: Shi Liang , Bischoff Kenneth M. , Kennelly Peter J. 
Published: December 2019

ABSTRACTA set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered aroundicfG, a gene implicated in the regulation of carbon metabolism, in the genome ofSynechocystissp. strain PCC 6803.slr1860is the ORF foricfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX fromBacillus subtilis. Bracketingslr1860/icfGare (i) ORFslr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases fromB. subtilis, and (ii) ORFsslr1856andslr1859, whose predicted products resemble the respective phosphoprotein substrates for theB. subtilisprotein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed inEscherichia colias a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORFslr1861encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.
An Iron-Regulated Alkyl Hydroperoxide Reductase (AhpC) Confers Aerotolerance and Oxidative Stress Resistance to the Microaerophilic Pathogen Campylobacter jejuni
Authors: Baillon Marie-Louise A. , van Vliet Arnoud H. M. , Ketley Julian M. , Constantinidou Chrystala , Penn Charles W. 
Published: December 2019

ABSTRACTMicroaerophiles likeCampylobacter jejunimust resist oxidative stresses during transmission or infection. Growth ofC. jejuni81116 under iron limitation greatly increased the expression of two polypeptides of 26 and 55 kDa. The identification of these proteins by N-terminal amino acid sequencing showed both to be involved in the defense against oxidative stress. The 55-kDa polypeptide was identical toC. jejunicatalase (KatA), whereas the N terminus of the 26-kDa polypeptide was homologous to a 26-kDaHelicobacter pyloriprotein. The gene encoding theC. jejuni26-kDa protein was cloned, and the encoded protein showed significant homology to the small subunit of alkyl hydroperoxide reductase (AhpC). The upstream region ofahpCencoded a divergent ferredoxin (fdxA) homolog, whereas downstream sequences containedflhBandmotBhomologs, which are involved in flagellar motility. There was no evidence for an adjacent homolog ofahpF, encoding the large subunit of alkyl hydroperoxide reductase. Reporter gene studies showed that iron regulation ofahpCandkatAis achieved at the transcriptional level. Insertional mutagenesis of theahpCgene resulted in an increased sensitivity to oxidative stresses caused by cumene hydroperoxide and exposure to atmospheric oxygen, while resistance to hydrogen peroxide was not affected. TheC. jejuniAhpC protein is an important determinant of the ability of this microaerophilic pathogen to survive oxidative and aerobic stress.
Antigen-43-Mediated Autoaggregation ofEscherichia coli Is Blocked by Fimbriation
Authors: Hasman Henrik , Chakraborty Trinad , Klemm Per 
Published: December 2019

ABSTRACT
Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli. Ag43 is responsible for the autoaggregation and flocculation of static liquid cultures of many E. colistrains. The expression of Ag43 has been reported to be phase variable and controlled by the product of the oxyR gene. Type 1 fimbriae are thin adhesive thread-like surface organelles responsible for bacterial receptor recognition and tissue colonization. Like that of Ag43, the expression of type 1 fimbriae is phase variable. Interestingly, previous results have suggested that the expression of type 1 fimbriae and the expression of Ag43 are mutually exclusive. In the present report, we show, by use of well-defined mutants, that fimbriation abolishes Ag43-mediated autoaggregation but does not affect Ag43 expression. Autoaggregation is shown to require an intercellular Ag43-Ag43 interaction, and the physical presence of fimbriae on the cells seems to abrogate this interaction. The Ag43 or OxyR status does not appear to influence fimbria expression, and our results suggest that the expression of Ag43 and the expression of fimbriae are independent processes.
The Periplasmic Murein Peptide-Binding Protein MppA Is a Negative Regulator of Multiple Antibiotic Resistance in Escherichia coli
Authors: Li HongShan , Park James T. 
Published: December 2019

ABSTRACTMppA is a periplasmic binding protein inEscherichia coliessential for uptake of the cell wall murein tripeptidel-alanyl-γ-d-glutamyl-meso-diaminopimelate. We have found serendipitously thatE. coliK-12 strains carrying a null mutation inmppAexhibit increased resistance to a wide spectrum of antibiotics and to cyclohexane. Normal sensitivity of themppAmutant to these agents is restored bymppAexpressed from a plasmid. As is observed in the multiple antibiotic resistance phenotype inE. colicells, themppAnull mutant overproduces the transcriptional activator, MarA, resulting in expression of the membrane-bound AcrAB proteins that function as a drug efflux pump. Reduced production of OmpF similar to that observed in the multiple antibiotic resistance phenotype is also seen in themppAmutant. These and other data reported herein indicate that MppA functions upstream of MarA in a signal transduction pathway to negatively regulate the expression ofmarAand hence of the MarA-driven multiple antibiotic resistance. Overproduction of cytoplasmic GadA and GadB and of several unidentified cytoplasmic membrane proteins as well as reduction in the amount of the outer membrane protein, OmpP, in themppAnull mutant indicate that MppA regulates a number of genes in addition to those already known to be controlled by MarA.
In Thermoanaerobacterium thermosulfurigenes EM1 S-Layer Homology Domains Do Not Attach to Peptidoglycan
Authors: Brechtel Elke , Bahl Hubert 
Published: December 2019

ABSTRACT
Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins). At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium. The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase. This enzyme was isolated fromT. thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli. The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall. In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T. thermosulfurigenes EM1 were included in these studies. The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi. Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall. Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria.