bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helicobacter pylori rocF
Is Required for Arginase Activity and Acid Protection In Vitro but Is Not Essential for Colonization of Mice or for Urease Activity
Authors: McGee David J. , Radcliff Fiona J. , Mendz George L. , Ferrero Richard L. , Mobley Harry L. T. 
Published: December 2019

ABSTRACT

Arginase of the
Helicobacter pylori
urea cycle hydrolyzes
l
-arginine to
l
-ornithine and urea.
H. pylori
urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in
H. pylori
nitrogen metabolism. The roles of arginase in the physiology of
H. pylori
were investigated in vitro and in vivo, since arginase in
H. pylori
is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The
H. pylori
gene
hp1399
, which is orthologous to the
Bacillus subtilis rocF
gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three
H. pylori
strains were made by using two different constructs: 236-2 and
rocF::aphA3
. In contrast to wild-type (WT) strains, all
rocF
mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of
H. pylori
, the
rocF::aphA3
mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the
rocF::aphA3
mutant was not reversed by the addition of
l
-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that
rocF
is not required for urease activity in vitro. Finally,
H. pylori
mouse-adapted strain SS1 and the 236-2
rocF
isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the
rocF::aphA3
mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of
H. pylori
recovered from these mice (in log
10
CFU) were 6.1, 5.5, and 4.1, respectively. Thus,
H. pylori rocF
is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.
Protection against 3′-to-5′ RNA Decay in
Bacillus subtilis
Authors: Farr Glen A. , Oussenko Irina A. , Bechhofer David H. 
Published: December 2019

ABSTRACT

A 320-nucleotide RNA with several characteristic features was expressed in
Bacillus subtilis
to study RNA processing. The RNA consisted of a 5′-proximal sequence from bacteriophage SP82 containing strong secondary structure, a Bs-RNase III cleavage site, and the 3′-proximal end of the
ermC
transcriptional unit. Comparison of RNA processing in a wild-type strain and a strain in which the
pnpA
gene, coding for polynucleotide phosphorylase (PNPase), was deleted, as well as in vitro assays of phosphate-dependent degradation, showed that PNPase activity could be stalled in vivo and in vitro. Analysis of mutations in the SP82 moiety mapped the block to PNPase processivity to a particular stem-loop structure. This structure did not provide a block to processivity in the
pnpA
strain, suggesting that it was specific for PNPase. An abundant RNA with a 3′ end located in the
ermC
coding sequence was detected in the
pnpA
strain but not in the wild type, indicating that this block is specific for a different 3′-to-5′ exonuclease. The finding of impediments to 3′-to-5′ degradation, with specificities for different exonucleases, suggests the existence of discrete intermediates in the mRNA decay pathway.
Sliding Motility in Mycobacteria
Authors: Martínez Asunción , Torello Sandra , Kolter Roberto 
Published: December 2019

ABSTRACT

Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here that
Mycobacterium smegmatis
can spread on the surface of growth medium by a sliding mechanism.
M. smegmatis
spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to
M. smegmatis
but is also a property of the slow-growing opportunistic pathogen
M. avium
. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host.
Cellobiose-6-Phosphate Hydrolase (CelF) of
Escherichia coli
: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases
Authors: Thompson John , Ruvinov Sergei B. , Freedberg Darón I. , Hall Barry G. 
Published: December 2019

ABSTRACT

The gene
celF
of the cryptic
cel
operon of
Escherichia coli
has been cloned, and the encoded 6-phospho-β-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC
3.2.1.86
]) has been expressed and purified in a catalytically active state. Among phospho-β-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD
+
for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-β-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-β-glucoside-6P, and the chromogenic analog,
p
-nitrophenyl-β-
d
-glucopyranoside-6P. In the absence of a metal ion and NAD
+
, purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD
+
appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (
M
w
, ∼200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (
M
r
, ∼50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues;
M
r
= 50,512). Comparative sequence alignments provide tentative identification of the NAD
+
-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu
112
and Glu
356
) of CelF.
The Basis of Ammonium Release in
nifL
Mutants of
Azotobacter vinelandii
Authors: Brewin Brett , Woodley Paul , Drummond Martin 
Published: December 2019

ABSTRACT

In
Azotobacter vinelandii
, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by
nifLA
. Certain mutations in
nifL
result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of
nif
gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination of
nifL
or overexpression of
nifA
, resulted in ammonium release, correlating with enhanced levels of
nifH
mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH
4
+
uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.
Chromate Efflux by Means of the ChrA Chromate Resistance Protein from
Pseudomonas aeruginosa
Authors: Alvarez Angel H. , Moreno-Sánchez Rafael , Cervantes Carlos 
Published: December 2019

ABSTRACT

Everted membrane vesicles of
Pseudomonas aeruginosa
PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more
51
CrO
4
2−
than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent

K
m

of 0.12 mM chromate and a
V
max
of 0.5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.
Gene Disruption through Homologous Recombination in
Spiroplasma citri
: an
scm1
-Disrupted Motility Mutant Is Pathogenic
Authors: Duret Sybille , Danet Jean-Luc , Garnier Monique , Renaudin Joël 
Published: December 2019

ABSTRACT

To determine whether homologous recombination could be used to inactivate selected genes in
Spiroplasma citri
, plasmid constructs were designed to disrupt the motility gene
scm1
. An internal
scm1
gene fragment was inserted into plasmid pKT1, which replicates in
Escherichia coli
but not in
S. citri
, and into the
S. citri oriC
plasmid pBOT1, which replicates in spiroplasma cells as well as in
E. coli
. Electrotransformation of
S. citri
with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the
oriC
region or at the
scm1
gene. In the latter case, plasmid integration by a single crossover between the
scm1
gene fragment carried by the plasmid and the full-length
scm1
gene carried by the chromosome led to a nonmotile phenotype. Transmission of the
scm1
-disrupted mutant to periwinkle (
Catharanthus roseus
) plants through injection into the leafhopper vector (
Circulifer haematoceps
) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type
S. citri
strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the
S. citri oriC
plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in
S. citri
, a mollicute which probably lacks a functional RecA protein.
A Cyclic AMP Receptor Protein Mutant That Constitutively Activates an
Escherichia coli
Promoter Disrupted by an IS
5
Insertion
Authors: Podolny Vladimir , Lin E. C. C. , Hochschild Ann 
Published: December 2019

ABSTRACT

Previously an
Escherichia coli
mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the
fucO
gene (in the
fucAO
operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS
5
insertion in the intergenic regulatory region between the divergently oriented
fucAO
and
fucPIK
operons. Though expression of the
fucAO
operon was constitutive, the
fucPIK
operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the
crp
gene was selected. Here we identify this
crp
mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS
5
-disrupted and the wild-type
fucPIK
promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the
fucPIK
promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS
5
-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.
Type 1 Fimbriation and Phase Switching in a Natural
Escherichia coli fimB
Null Strain, Nissle 1917
Authors: Stentebjerg-Olesen Bodil , Chakraborty Trinad , Klemm Per 
Published: December 2019

ABSTRACT

Escherichia coli
Nissle 1917 has been used as a probiotic against intestinal disorders for many decades. It is a good colonizer of the human gut and has been reported to be able to express type 1 fimbriae. Type 1 fimbriae are surface organelles which mediate α-
d
-mannose-sensitive binding to various host cell surfaces. The expression is phase variable, and two tyrosine recombinases, FimB and FimE, mediate the inversion of the fimbrial phase switch. Current evidence suggests that FimB can carry out recombination in both directions, whereas FimE-catalyzed switching is on to off only. We show here that under liquid shaking growth conditions, Nissle 1917 did not express type 1 fimbriae, due to a truncation of the
fimB
gene by an 1,885-bp insertion element. Despite its
fimB
null status, Nissle 1917 was still capable of off-to-on switching of the phase switch and expressing type 1 fimbriae when grown under static conditions. This phase switching was not catalyzed by FimE, by truncated FimB, or by information residing within the insertion element. No further copies of
fimB
seemed to be present on the chromosome of Nissle 1917, suggesting that another tyrosine recombinase in Nissle 1917 is responsible for the low-frequency off-to-on inversion of the phase switch that is strongly favored under static growth conditions. This is the first report documenting the non-FimB- or non-FimE-catalyzed inversion of the
fim
switch.
Multiple Control of Flagellum Biosynthesis in
Escherichia coli
: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the
flhDC
Master Operon
Authors: Soutourina O. , Kolb A. , Krin E. , Laurent-Winter C. , Rimsky S. , Danchin A. , Bertin P. 
Published: December 2019

ABSTRACT

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that
crp
and
hns
mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of
fliA
and
fliC
, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the
flhDC
master operon restored, at least in part, motility in
crp
and
hns
mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates
flhDC
expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the
flhDC
promoter was decreased in the
crp
strain and increased in the
hns
mutant. In contrast, the activity of a transcriptional fusion encompassing the entire
flhDC
regulatory region extending to the ATG translational start codon was strongly reduced in both
hns
and
crp
mutants. These results suggest that the region downstream of the +1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a
crp
mutant by activation-deficient CAP mutated proteins and characterization of
cfs
, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates
flhDC
transcription by binding to its promoter and interacting with RNA polymerase.
CDC64
Encodes Cytoplasmic Alanyl-tRNA Synthetase, Ala1p, of
Saccharomyces cerevisiae
Authors: Wrobel Carolyn , Schmidt Emmett V. , Polymenis Michael 
Published: December 2019

ABSTRACT

The
cdc64-1
mutation causes G
1
arrest in
Saccharomyces cerevisiae
corresponding to a type II Start phenotype. We report that
CDC64
encodes Ala1p, an alanyl-tRNA synthetase. Thus,
cdc64-1
might affect charging of tRNA
Ala
and thereby initiation of cell division.
Structural and Functional Analyses of the Secondary Cell Wall Polymer of
Bacillus sphaericus
CCM 2177 That Serves as an S-Layer-Specific Anchor
Authors: Ilk Nicola , Kosma Paul , Puchberger Michael , Egelseer Eva M. , Mayer Harald F. , Sleytr Uwe B. , Sára Margit 
Published: December 2019

ABSTRACT

Sacculi of
Bacillus sphaericus
CCM 2177 contain a secondary cell wall polymer which was completely extracted with 48% hydrofluoric acid. Nuclear magnetic resonance analysis showed that the polymer is composed of repeating units, as follows: →3)-[4,6-
O
-(1-carboxyethylidene)]
∼0.5
-β-
d
-Man
p
NAc-(1→4)-β-
d
-Glc
p
NAc-(1→. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.
Characterization of IS
1547
, a New Member of the IS
900
Family in the
Mycobacterium tuberculosis
Complex, and Its Association with IS
6110
Authors: Fang Z. , Doig C. , Morrison N. , Watt B. , Forbes K. J. 
Published: December 2019

ABSTRACT

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS
116
, IS
900
, IS
901
, and IS
1110
have been proposed as members of the IS
900
family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS
1547
, which was first identified in a clinical isolate of
Mycobacterium tuberculosis
. Its structure, insertion site, and putative transposase all conform with the conventions of the IS
900
family, suggesting that it is a new member of this family. IS
1547
was detected only in isolates of the
M. tuberculosis
complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the
M. tuberculosis
complex and for distinguishing different strains of
M. tuberculosis
.
ipl
is a preferential locus for IS
6110
insertion where there are eight known different insertion sites for IS
6110
. Surprisingly, the DNA sequence of
ipl
is now known to be a part of IS
1547
, meaning that IS
1547
is a preferential site for IS
6110
insertion.
Sporadic Distribution of tRNA
CCU
Arg
Introns among α-Purple Bacteria: Evidence for Horizontal Transmission and Transposition of a Group I Intron
Authors: Paquin Bruno , Heinfling Annette , Shub David A. 
Published: December 2019

ABSTRACT

A group I intron interrupts the tRNA
CCU
Arg
gene of the α-purple bacterium
Agrobacterium tumefaciens
(B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173–176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of α-purple bacteria. Of 10 newly identified tRNA
CCU
Arg
genes, we found only two that contained an intron homologous to that of the
Agrobacterium
tRNA
CCU
Arg
intron. This restricted and scattered distribution of the tRNA
CCU
Arg
intron among α-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA
UAA
Leu
introns found in strains of the cyanobacterium
Microcystis aeruginosa
(K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293–298, 1997) and α-purple tRNA
CCU
Arg
introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA
UAA
Leu
introns.
The
merG
Gene Product Is Involved in Phenylmercury Resistance in
Pseudomonas
Strain K-62
Authors: Kiyono Masako , Pan-Hou Hidemitsu 
Published: December 2019

ABSTRACT

The physiological function of a new gene, hereby designated
merG
, located between
merA
and
merB
on the broad-spectrum
mer
operon of
Pseudomonas
strain K-62 plasmid pMR26 was investigated. The 654-bp
merG
gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the
merG
gene from the broad-spectrum
mer
operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain.
Escherichia coli
cells bearing pMU29, which carries a deletion of the
merG
gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the
merG
gene in a compatible plasmid was transformed into the
E. coli
strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the
merG
gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.
Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of
Nocardioides
sp. Strain KP7
Authors: Adachi Kyoko , Iwabuchi Tokuro , Sano Hiroshi , Harayama Shigeaki 
Published: December 2019

ABSTRACT

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in
Nocardioides
sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (
E
)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name,
trans
-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by
1
H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.