bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated
Escherichia coli
: Requirement for DNA End Processing
Authors: Thoms Brigitte , Wackernagel Wilfried 
Published: December 2019

ABSTRACT

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m
2
). The activity decrease was seen by an increase in survival of phage T4
2
−
of about 200-fold (phage T4
2
−
has defective duplex DNA end-protecting gene
2
protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with
ssb
+
) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in
xonA
,
recJ
, and
sbcCD
, particularly in the
recJ sbcCD
and
xonA recJ sbcCD
combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.
Regulation of Motility Behavior in
Myxococcus xanthus
May Require an Extracytoplasmic-Function Sigma Factor
Authors: Ward Mandy J. , Lew Helen , Treuner-Lange Anke , Zusman David R. 
Published: December 2019

ABSTRACT

Using interaction trap technology, we identified a putative extracytoplasmic-function (ECF) sigma factor (RpoE1) in
Myxococcus xanthus
, a bacterium which has a complex life cycle that includes fruiting body formation. The first domain of the response regulator protein FrzZ, a component of the Frz signal transduction system, was used as bait. Although the RpoE1 protein displayed no interactions with control proteins presented as bait, a weak interaction with a second
M. xanthus
response regulator (AsgA) was observed. While the specificity of the FrzZ-RpoE1 interaction therefore remains speculative, cloning and sequencing of the region surrounding
rpoE1
localized it to a position downstream of the
frzZ
gene. A potential promoter site for binding of an ECF sigma factor was identified upstream of
rpoE1
, suggesting the gene may be autoregulated. However, primer extension studies suggested that transcription of
rpoE1
occurs under both vegetative and developmental conditions from a ς
70
-like promoter. Dot blot analysis of RNA preparations confirmed the low-level, constitutive expression of
rpoE1
during both stages of the life cycle. Analysis of an insertion mutant also indicated a role for RpoE1 under both vegetative and developmental conditions, since swarming was reduced on nutrient-rich agar and developmental aggregation was effected under starvation conditions, especially at high cell densities. An insertion mutation introduced into the gene directly downstream of
rpoE1
(
orf5
) did not result in either swarming or developmental aggregation defects, even though the gene is transcribed as part of the same operon. Therefore, we propose that this new ECF sigma factor could play a role in the transcriptional regulation of genes involved in motility behavior during both stages of the complex
M. xanthus
life cycle.
Role of the
Escherichia coli
SurA Protein in Stationary-Phase Survival
Authors: Lazar Sara W. , Almirón Marta , Tormo Antonio , Kolter Roberto 
Published: December 2019

ABSTRACT

SurA is a periplasmic peptidyl-prolyl isomerase required for the efficient folding of extracytoplasmic proteins. Although the
surA
gene had been identified in a screen for mutants that failed to survive in stationary phase, the role played by SurA in stationary-phase survival remained unknown. The results presented here demonstrate that the survival defect of
surA
mutants is due to their inability to grow at elevated pH in the absence of ς
S
. When cultures of
Escherichia coli
were grown in peptide-rich Luria-Bertani medium, the majority of the cells lost viability during the first two to three days of incubation in stationary phase as the pH rose to pH 9. At this time the surviving cells resumed growth. In cultures of
surA rpoS
double mutants the survivors lysed as they attempted to resume growth at the elevated pH. Cells lacking penicillin binding protein 3 and ς
S
had a survival defect similar to that of
surA rpoS
double mutants, suggesting that SurA foldase activity is important for the proper assembly of the cell wall-synthesizing apparatus.
Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs
Authors: Slupska Malgorzata M. , King Angela G. , Lu Louise I. , Lin Rose H. , Mao Emily F. , Lackey Chantal A. , Chiang Ju-Huei , Baikalov Claudia , Miller Jeffrey H. 
Published: December 2019

ABSTRACT

We previously described
Escherichia coli
mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, ɛ, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered ɛ subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of
mutD
alleles. In the present work, we extended the study of specific
mutD
alleles and constructed 16 altered
mutD
genes by replacing each aspartic acid codon, in series, with a glycine codon in the
dnaQ
gene that encodes ɛ. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the
mutD
gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the ɛ subunit playing a major role in the mutator effect of misreading tRNAs.
CHR, a Novel Family of Prokaryotic Proton Motive Force-Driven Transporters Probably Containing Chromate/Sulfate Antiporters
Authors: Nies Dietrich H. , Koch Sandra , Wachi Shinichiro , Peitzsch Nicola , Saier Milton H. 
Published: December 2019

ABSTRACT
We describe a small family of proteins, CHR, which contains members that function in chromate and/or sulfate transport. CHR proteins occur in bacteria and archaea. They consist of about 400 amino acyl residues, appear to have 10 transmembrane α-helical segments in an unusual 4+6 arrangement, and arose by an intragenic duplication event.
Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from
Mycobacterium smegmatis
Authors: Boshoff Helena I. M. , Mizrahi Valerie 
Published: December 2019

ABSTRACT

The pyrazinamidase from
Mycobacterium smegmatis
was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of
M. smegmatis
. An open reading frame, designated
pzaA
, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the
pzaA
gene by homologous recombination yielded a mutant,
pzaA
::
aph
, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of
M. smegmatis
. Recombinant forms of the
M. smegmatis
PzaA and the
Mycobacterium tuberculosis
pyrazinamidase/nicotinamidase (PncA) were produced in
Escherichia coli
and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable

K
m

values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for
M. tuberculosis
was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of
pzaA
conferred PZA susceptibility on
M. smegmatis
by reducing the MIC of this drug to 150 μg/ml.
Identification and Characterization of the
fis
Operon in Enteric Bacteria
Authors: Beach Michael B. , Osuna Robert 
Published: December 2019

ABSTRACT

The small DNA binding protein Fis is involved in several different biological processes in
Escherichia coli
. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at
oriC. fis
has also been isolated from
Salmonella typhimurium
, and the genomic sequence of
Haemophilus influenzae
reveals its presence in this bacteria. This work extends the characterization of
fis
to other organisms. Very similar
fis
operon structures were identified in the enteric bacteria
Klebsiella pneumoniae
,
Serratia marcescens
,
Erwinia carotovora
, and
Proteus vulgaris
but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in
K. pneumoniae
,
S. marcescens
,
E. coli
, and
S. typhimurium
and 96 to 98% identical when
E. carotovora
and
P. vulgaris
Fis are considered. The deduced amino acid sequence for
H. influenzae
Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the
E. carotovora
,
P. vulgaris
, and
H. influenzae
Fis proteins are not functionally identical. An open reading frame (ORF1) preceding
fis
in
E. coli
is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the
E. coli fis
P region from −53 to +27 and the region around −116 containing an
ihf
binding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.
Three Replicons of
Rhizobium
sp. Strain NGR234 Harbor Symbiotic Gene Sequences
Authors: Flores Margarita , Mavingui Patrick , Girard Lourdes , Perret Xavier , Broughton William J. , Martínez-Romero Esperanza , Dávila Guillermo , Palacios Rafael 
Published: December 2019

ABSTRACT

Rhizobium
sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234
a
, a megaplasmid (pNGR234
b
), and the chromosome. Symbiotic gene sequences not present in pNGR234
a
were analyzed by hybridization. DNA sequences homologous to the genes
fixLJKNOPQGHIS
were found on the chromosome, while sequences homologous to
nodPQ
and
exoBDFLK
were found on pNGR234
b
.
Complex Metabolic Phenotypes Caused by a Mutation in
yjgF
, Encoding a Member of the Highly Conserved YER057c/YjgF Family of Proteins
Authors: Enos-Berlage Jodi L. , Langendorf Mark J. , Downs Diana M. 
Published: December 2019

ABSTRACT

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in
Salmonella typhimurium
. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in
yjgF
, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of
yjgF
mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.
The First Gene of theBacillus subtilis clpCOperon,ctsR, Encodes a Negative Regulator of Its Own Operon and Other Class III Heat Shock Genes
Authors: Krüger Elke , Hecker Michael 
Published: December 2019

ABSTRACTTheBacillus subtilis clpCoperon is regulated by two stress induction pathways relying on either ςBor a class III stress induction mechanism acting at a ςA-like promoter. When theclpCoperon was placed under the control of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Pspacpromoter, dramatic repression of the naturalclpCpromoters fused to alacZreporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of theclpCoperon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of theclpCoperon,ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion ofctsRabolished the negative regulation and resulted in high expression of both theclpCoperon and theclpPgene under nonstressed conditions. Nevertheless, a further increase inclpCandclpPmRNA levels was observed after heat shock, even in the absence of ςB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by thectsRdeletion. Studies with differentclpCpromoter fragments either fused to the reporter genebgaBor used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of theclpCoperon, as well as other class III heat shock genes, by preventing unstressed transcription from either the ςB- or ςA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.
Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria
Authors: Thulasiraman Padmamalini , Newton Salete M. C. , Xu Jide , Raymond Kenneth N. , Mai Christine , Hall Angela , Montague Marjorie A. , Klebba Phillip E. 
Published: December 2019

ABSTRACT

The ligand-gated outer membrane porin FepA serves
Escherichia coli
as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their
59
Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (

K
d

≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K
3
MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from
Bordetella bronchisepticus
,
Pseudomonas aeruginosa
, and
Salmonella typhimurium
in the native organisms and as plasmid-mediated clones expressed in
E. coli
. All the transport proteins bound ferric enterobactin with high affinity (

K
d

≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10
9
cells) in their own particular membrane environments. However, the FepA and IroN proteins of
S. typhimurium
failed to efficiently function in
E. coli
. For
E. coli
,
S. typhimurium
, and
P. aeruginosa
, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.
Sucrose-Phosphate Synthase from
Synechocystis
sp. Strain PCC 6803: Identification of the
spsA
Gene and Characterization of the Enzyme Expressed in
Escherichia coli
Authors: Curatti Leonardo , Folco Eduardo , Desplats Paula , Abratti Gustavo , Limones Verónica , Herrera-Estrella Luis , Salerno Graciela 
Published: December 2019

ABSTRACT

The first identification and characterization of a prokaryotic gene (
spsA
) encoding sucrose-phosphate synthase (SPS) is reported for
Synechocystis
sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.
The Fur-Regulated Gene Encoding the Alternative Sigma Factor PvdS Is Required for Iron-Dependent Expression of the LysR-Type Regulator PtxR in
Pseudomonas aeruginosa
Authors: Vasil Michael L. , Ochsner Urs A. , Johnson Zaiga , Colmer Jane A. , Hamood Abdul N. 
Published: December 2019

ABSTRACT

We previously identified a novel regulator of the exotoxin A gene (
toxA
) in
Pseudomonas aeruginosa
, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in
P. aeruginosa
. Because
toxA
expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of
ptxR
itself in the context of these regulators was examined. RNase protection analyses of
ptxR
transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a
fur
mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of
ptxR
in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of
ptxR
. Iron-regulated transcription of
ptxR
from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene
pvdS
, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of
ptxR
. The iron-regulated expression of
ptxR
under microaerobic conditions from the P2 promoter of
ptxR
is mediated indirectly by Fur through the iron-regulated expression of
pvdS
. In contrast,
pvdS
-mediated iron regulation of
ptxR
under aerobic conditions is Fur independent.
Purine Salvage in Two Halophilic Archaea: Characterization of Salvage Pathways and Isolation of Mutants Resistant to Purine Analogs
Authors: Stuer-Lauridsen Birgitte , Nygaard Per 
Published: December 2019

ABSTRACT

In exponentially growing cultures of the extreme halophile
Halobacterium halobium
and the moderate halophile
Haloferax volcanii
, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed. This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-α-1-pyrophosphate) in archaea. The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays. The levels varied significantly between the two organisms. The most significant difference was the absence of GMP reductase activity in
H. halobium
. The metabolism of exogenous purines was investigated in growing cultures. Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids. Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis.
H. volcanii
was more sensitive than
H. halobium
, and purine base analogs were more toxic than nucleoside analogs. Growth of
H. volcanii
was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of
H. halobium
. Spontaneous mutants resistant to purine analogs were isolated. The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake. A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine. The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels. Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.
Mutational Analysis of UMP Kinase from
Escherichia coli
Authors: Bucurenci Nadia , Serina Lidia , Zaharia Cristina , Landais Stéphanie , Danchin Antoine , Bârzu Octavian 
Published: December 2019

ABSTRACT

UMP kinase from
Escherichia coli
is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of
E. coli
UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).