bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Analysis of the rnc-era-recOOperon from Pseudomonas aeruginosa
Authors: Powell Bradford , Peters Howard K. , Nakamura Yoshikazu , Court Donald 
Published: December 2019

ABSTRACT
The rnc operon from Pseudomonas aeruginosahas been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of thernc, era, and recO genes inEscherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of eramutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.
Reduction of Adenosine-5′-Phosphosulfate Instead of 3′-Phosphoadenosine-5′-Phosphosulfate in Cysteine Biosynthesis by
Rhizobium meliloti
and Other Members of the Family
Rhizobiaceae
Authors: Abola A. Pia , Willits Michael G. , Wang Richard C. , Long Sharon R. 
Published: December 2019

ABSTRACT

We have cloned and sequenced three genes from
Rhizobium meliloti
(
Sinorhizobium meliloti
) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the
Escherichia coli cysDN
genes, which code for an ATP sulfurylase (EC
2.7.7.4
). The third gene has homology to the
E. coli cysH
gene, a 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase (EC
1.8.99.4
), but has greater homology to a set of genes found in
Arabidopsis thaliana
that encode an adenosine-5′-phosphosulfate (APS) reductase. In order to determine the specificity of the
R. meliloti
reductase, the
R. meliloti cysH
homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the
A. thaliana
reductases, the histidine-tagged
R. meliloti cysH
gene product appears to favor APS over PAPS as a substrate, with a

K
m

for APS of 3 to 4 μM but a

K
m

for PAPS of >100 μM. In order to determine whether this preference for APS is unique to
R. meliloti
among members of the family
Rhizobiaceae
or is more widespread, cell extracts from
R. leguminosarum
,
Rhizobium
sp. strain NGR234,
Rhizobium fredii
(
Sinorhizobium fredii
), and
Agrobacterium tumefaciens
were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.
The
napF
and
narG
Nitrate Reductase Operons in
Escherichia coli
Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite
Authors: Wang Henian , Tseng Ching-Ping , Gunsalus Robert P. 
Published: December 2019

ABSTRACT

Escherichia coli
synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the
narGHJI
operon and a periplasmic cytochrome
c
-linked nitrate reductase encoded by the
napFDAGHBC
operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine
napF
and
narG
gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the
napF-lacZ
and
narG-lacZ
reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The
napF
operon apparently encodes a “low-substrate-induced” reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the
narGHJI
operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The
narGHJI
operon is therefore a “high-substrate-induced” operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of
napF
gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of
narG
and
napF
expression must be reconsidered.
A New Class of Cobalamin Transport Mutants (
btuF
) Provides Genetic Evidence for a Periplasmic Binding Protein in
Salmonella typhimurium
Authors: Van Bibber Michael , Bradbeer Clive , Clark Nica , Roth John R. 
Published: December 2019

ABSTRACT

No periplasmic binding protein has been demonstrated for the ATP-binding cassette (ABC)-type cobalamin transporter BtuCD. New mutations (
btuF
) are described that affect inner-membrane transport. The BtuF protein has a signal sequence and resembles the periplasmic binding proteins of several other ABC transporters.
Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in
Escherichia coli
K-12
Authors: Atlung T. , Hansen F. G. 
Published: December 2019

ABSTRACT

Expression of the
dnaA
gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14°C than at 37°C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30°C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of
dnaA
gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.
Involvement of the
cis/trans
Isomerase Cti in Solvent Resistance of
Pseudomonas putida
DOT-T1E
Authors: Junker Frank , Ramos Juan L. 
Published: December 2019

ABSTRACT

Pseudomonas putida
DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the
cti
gene of this strain, which encodes the
cis/trans
isomerase, termed Cti, that catalyzes the
cis-trans
isomerization of esterified fatty acids in phospholipids, mainly
cis
-oleic acid (C
16:1,9
) and
cis
-vaccenic acid (C
18:1,11
), in response to solvents. To determine the importance of this
cis/trans
isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5°C, the Cti-null mutant strain grew significantly slower at temperatures above 37°C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the
cti
gene is present in all the tested
P. putida
strains, including non-solvent-resistant ones, and in some other
Pseudomonas
species.
Analysis of Fusion Junctions of Circularized Chromosomes in
Streptomyces griseus
Authors: Kameoka Daisuke , Lezhava Alexander , Zenitani Hiroyuki , Hiratsu Keiichiro , Kawamoto Makoto , Goshi Kohei , Inada Kuninobu , Shinkawa Hidenori , Kinashi Haruyasu 
Published: December 2019

ABSTRACT

A filamentous soil bacterium,
Streptomyces griseus
2247, carries a 7.8-Mb linear chromosome. We previously showed by macrorestriction analysis that mutagenic treatments easily caused deletions at both ends of its linear chromosome and changed the chromosome to a circular form. In this study, we confirmed chromosomal circularization by cloning and sequencing the junction fragments from two deletion mutants, 404-23 and N2. The junction sequences were compared with the corresponding right and left deletion end sequences in the parent strain, 2247. No homology and a 6-bp microhomology were found between the two deletion ends of the 404-23 and N2 mutants, respectively, which indicate that the chromosomal circularization was caused by illegitimate recombination without concomitant amplification. The circularized chromosomes were stably maintained in both mutants. Therefore, the chromosomal circularization might have occurred to prevent lethal deletions, which otherwise would progress into the indispensable central regions of the chromosome.
The
Caulobacter crescentus
CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties
Authors: Lin Bin , Covalle Kelly L. , Maddock Janine R. 
Published: December 2019

ABSTRACT

The
Caulobacter crescentus
CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30°C and displayed equilibrium binding constants of 1.2 and 0.5 μM, respectively, in the presence of Mg
2+
. In the absence of Mg
2+
, the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively.
N
-Methyl-3′-
O
-anthranoyl (mant)–guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg
2+
was extremely rapid (

k
d

= 1.4 and 1.5 s
−1
, respectively), 10
3
- to 10
5
-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg
2+
. Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 × 10
−4
s
−1
corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.
A Sequence Downstream of the Initiation Codon Is Essential for Cold Shock Induction of
cspB
of
Escherichia coli
Authors: Etchegaray Jean-Pierre , Inouye Masayori 
Published: December 2019

ABSTRACT

Cold shock induction of
cspB
has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of
cspB
at low temperature also requires the translational
cis
-acting element called the downstream box (DB). Full induction of
cspB
at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.
Role of CCAA Nucleotide Repeats in Regulation of Hemoglobin and Hemoglobin-Haptoglobin Binding Protein Genes of
Haemophilus influenzae
Authors: Ren Zhen , Jin Hongfan , Whitby Paul W. , Morton Daniel J. , Stull Terrence L. 
Published: December 2019

ABSTRACT

Haemophilus influenzae
utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The
H. influenzae
hemoglobin- and hemoglobin-haptoglobin binding protein genes,
hgpA
,
hgpB
, and
hgpC
, contain lengths of tetrameric CCAA repeats. Using an
hgpA-lacZ
translational gene fusion, we demonstrate phase-variable expression of
lacZ
associated with alteration in the length of the CCAA repeat region.
Expression of the
Staphylococcus aureus
UDP-
N
-Acetylmuramoyl-
l
-Alanyl-
d
-Glutamate:
l
-Lysine Ligase in
Escherichia coli
and Effects on Peptidoglycan Biosynthesis and Cell Growth
Authors: Mengin-Lecreulx Dominique , Falla Tim , Blanot Didier , van Heijenoort Jean , Adams David J. , Chopra Ian 
Published: December 2019

ABSTRACT

The monomer units in the
Escherichia coli
and
Staphylococcus aureus
cell wall peptidoglycans differ in the nature of the third amino acid in the
l
-alanyl-γ-
d
-glutamyl-X-
d
-alanyl-
d
-alanine side chain, where X is
meso
-diaminopimelic acid or
l
-lysine, respectively. The
murE
gene from
S. aureus
encoding the UDP-
N
-acetylmuramoyl-
l
-alanyl-
d
-glutamate:
l
-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in
E. coli
rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the
meso
-diaminopimelic acid residues were replaced by
l
-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively
meso
-diaminopimelic acid, suggesting that no transpeptidation could occur between the ɛ-amino group of
l
-lysine and the α-carboxyl group of
d
-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of
meso
-diaminopimelic acid- and
l
-lysine-containing peptidoglycan precursors confirmed the presence of
l
-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.
Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential
uppS
Gene
Authors: Apfel Christian M. , Takács Béla , Fountoulakis Michael , Stieger Martin , Keck Wolfgang 
Published: December 2019

ABSTRACT

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-
trans
,poly-
cis
-decaprenylcistransferase; EC
2.5.1.31
) was purified from the soluble fraction of
Escherichia coli
by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (
E
,
E
)-[1-
3
H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all
E. coli
proteins yielded a single match with a protein of unassigned function (SWISS-PROT
Q47675
; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in
Saccharomyces cerevisiae
, and in
Caenorhabditis elegans
. The homologous genes (
uppS
) were cloned from
E. coli
,
Haemophilus influenzae
, and
Streptococcus pneumoniae
, expressed in
E. coli
as amino-terminal His-tagged fusion proteins, and purified over a Ni
2+
affinity column. An untagged version of the
E. coli uppS
gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged
E. coli
Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl
2
dependent. With the use of a regulatable gene disruption system, we demonstrated that
uppS
is essential for growth in
S. pneumoniae
R6.
A Second Operator Is Involved in
Pseudomonas aeruginosa
Elastase (
lasB
) Activation
Authors: Anderson Ronda M. , Zimprich Chad A. , Rust Lynn 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
LasB elastase gene (
lasB
) transcription is controlled by the two-component quorum-sensing system of LasR, and the autoinducer, 3OC
12
-HSL (
N
-3-[oxododecanoyl]homoserine lactone). LasR and 3OC
12
-HSL-mediated
lasB
activation requires a functional operator sequence (OP1) in the
lasB
promoter region. Optimal activation of
lasB
, however, requires a second sequence of 70% identity to OP1, named OP2, located 43 bp upstream of OP1. In this study, we used sequence substitutions and insertion mutations in
lasB
p-
lacZ
fusion plasmids to explore the role of OP2 in
lasB
activation. Our results demonstrate that (i) OP1 and OP2 synergistically mediate
lasB
activation; (ii) OP2, like OP1, responds to LasR and 3OC
12
-HSL; and (iii) the putative autoinducer-binding domain of LasR is not required for synergistic activation from OP1 and OP2.
Induction of β-Lactamase Influences the Course of Development in
Myxococcus xanthus
Authors: O’Connor Kathleen A. , Zusman David R. 
Published: December 2019

ABSTRACT

Myxococcus xanthus
is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of β-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O’Connor and D. R. Zusman, Mol. Microbiol. 24:839–850, 1997). In this paper, we show that the chromosomally encoded β-lactamase of
M. xanthus
is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of β-lactamase in
M. xanthus
, such as ampicillin,
d
-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of β-lactamase allows
M. xanthus
to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of β-lactamase is an integral step in the development of
M. xanthus
and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of β-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of β-lactamase plays an important role in aggregation and sporulation in
M. xanthus
.
Ferric Enterochelin Transport in
Yersinia enterocolitica
: Molecular and Evolutionary Aspects
Authors: Schubert S. , Fischer D. , Heesemann J. 
Published: December 2019

ABSTRACT

Yersinia enterocolitica
is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the
Yersinia fep-fes
gene cluster orthologous to the
Escherichia coli
ferrienterochelin transport genes (
fepA
,
fepDGC
, and
fepB
) and the esterase gene
fes
. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by
fepC
and
fes
, respectively. A frameshift mutation within the
fepA
gene led to expression of a truncated polypeptide of 40 kDa. The
fepD
,
fepG
, and
fes
genes of
Y. enterocolitica
were shown to complement corresponding
E. coli
mutants. Insertional mutagenesis of
fepD
or
fes
genes abrogates enterochelin-supported growth of
Y. enterocolitica
on iron-chelated media. In contrast to
E. coli
, the
fep-fes
gene cluster in
Y. enterocolitica
consists solely of genes required for uptake and utilization of enterochelin (
fep
) and not of enterochelin synthesis genes such as
entF
. By Southern hybridization,
fepDGC
and
fes
sequences could be detected in
Y. enterocolitica
biotypes IB, IA, and II but not in biotype IV strains,
Yersinia pestis
, and
Yersinia pseudotuberculosis
strains. According to sequence alignment data and the coherent structure of the
Yersinia fep-fes
gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family
Enterobacteriaceae
.
Initial Reactions in Anaerobic Oxidation of
m
-Xylene by the Denitrifying Bacterium
Azoarcus
sp. Strain T
Authors: Krieger Cynthia J. , Beller Harry R. , Reinhard Martin , Spormann Alfred M. 
Published: December 2019

ABSTRACT

The initial enzymatic steps in anaerobic
m
-xylene oxidation were studied in
Azoarcus
sp. strain T, a denitrifying bacterium capable of mineralizing
m
-xylene via 3-methylbenzoate. Permeabilized cells of
m
-xylene-grown
Azoarcus
sp. strain T catalyzed the addition of
m
-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to
E
-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of
m
-xylene-grown
Azoarcus
sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo
m
-xylene consumption. Based on these findings, we propose that
Azoarcus
sp. strain T anaerobically oxidizes
m
-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and
E
-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.
Efficiency of Transcription from Promoter Sequence Variants in
Lactobacillus
Is Both Strain and Context Dependent
Authors: McCracken Andrea , Timms Peter 
Published: December 2019

ABSTRACT

The introduction of consensus −35 (TTGACA) and −10 (TATAAT) hexamers and a TG motif into the
Lactobacillus acidophilus
ATCC 4356 wild-type
slpA
promoter resulted in significant improvements (4.3-, 4.1-, and 10.7-fold, respectively) in transcriptional activity in
Lactobacillus fermentum
BR11. In contrast, the same changes resulted in decreased transcription in
Lactobacillus rhamnosus
GG. The TG motif was shown to be important in the context of weak −35 and −10 hexamers (
L. fermentum
BR11) or a consensus −10 hexamer (
L. rhamnosus
GG). Thus, both strain- and context-dependent effects are critical factors influencing transcription in
Lactobacillus.