bluuebunny/crossref_metadata_2025_split huggingface dataset:
SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions
Authors: Rahman M. Sayeedur , Humayun M. Zafri 
Published: December 2019

ABSTRACT

Escherichia coli
cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N
4
-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective
E. coli
strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous
lac
-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.
Role of Ribosome Release in Regulation of
tna
Operon Expression in
Escherichia coli
Authors: Konan Kouacou Vincent , Yanofsky Charles 
Published: December 2019

ABSTRACT

Expression of the degradative tryptophanase (
tna
) operon of
Escherichia coli
is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the
tna
operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region,
tnaC
, that contains a single, crucial, Trp codon. Studies with a
lacZ
reporter construct lacking the spacer region between
tnaC
and the first major structural gene,
tnaA
, suggested that tryptophan induction might involve
cis
action by the TnaC leader peptide on the ribosome translating the
tnaC
coding region. The leader peptide was hypothesized to inhibit ribosome release at the
tnaC
stop codon, thereby blocking Rho’s access to the transcript. Regulatory studies with deletion constructs of the
tna
operon of
Proteus vulgaris
supported this interpretation. In the present study the putative role of the
tnaC
stop codon in
tna
operon regulation in
E. coli
was examined further by replacing the natural
tnaC
stop codon, UGA, with UAG or UAA in a
tnaC
-stop codon-
tnaA′-′lacZ
reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in
E. coli
translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the
tnaC
stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1,
prfA1
, increased basal level expression 60-fold when the
tnaC
stop codon was UAG and 3-fold when this stop codon was UAA; basal level expression was reduced by 50% in the construct with the natural stop codon, UGA. In strains with any of the three stop codons and the
prfA1
mutation, the induced levels of
tna
operon expression were virtually identical. The effects of
tnaC
stop codon identity on expression were also examined in the absence of Rho action, using
tnaC
-stop codon-′
lacZ
constructs that lack the
tnaC-tnaA
spacer region. Expression was low in the absence of
tnaC
stop codon suppression. In most cases, tryptophan addition resulted in about 50% inhibition of expression when UGA was replaced by UAG or UAA and the appropriate suppressor was present. Introduction of the
prfA1
mutant allele increased expression of the suppressed construct with the UAG stop codon; tryptophan addition also resulted in ca. 50% inhibition. These findings provide additional evidence implicating the behavior of the ribosome translating
tnaC
in the regulation of
tna
operon expression.
The
Bradyrhizobium japonicum nolA
Gene Encodes Three Functionally Distinct Proteins
Authors: Loh John , Stacey Minviluz G. , Sadowsky Michael J. , Stacey Gary 
Published: December 2019

ABSTRACT

Examination of
nolA
revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA
1
) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA
2
and NolA
3
, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of
Bradyrhizobium japonicum
extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of
nolA
from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of
nolA
in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the
nolA
mutants to a promoterless
lacZ
yielded functional fusion proteins in both
Escherichia coli
and
B. japonicum
. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the
B. japonicum nod
genes. The expression of both NolA
2
and NolA
3
requires the presence of NolA
1
. NolA
1
or NolA
3
is required for the genotype-specific nodulation of soybean genotype PI 377578.
Characterization of an Insertion Sequence Element Associated with Genetically Diverse Plant Pathogenic
Streptomyces
spp
Authors: Healy Frank G. , Bukhalid Raghida A. , Loria Rosemary 
Published: December 2019

ABSTRACT

Streptomycetes are common soil inhabitants, yet few described species are plant pathogens. While the pathogenicity mechanisms remain unclear, previous work identified a gene,
nec1
, which encodes a putative pathogenicity or virulence factor.
nec1
and a neighboring transposase pseudogene, ORF
tnp
, are conserved among unrelated plant pathogens and absent from nonpathogens. The atypical GC content of
nec1
suggests that it was acquired through horizontal transfer events. Our investigation of the genetic organization of regions adjacent to the 3′ end of
nec1
in
Streptomyces scabies
84.34 identified a new insertion sequence (IS) element, IS
1629
, with homology to other IS elements from prokaryotic animal pathogens. IS
1629
is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase. Transposition of IS
1629
generates a 10-bp target site duplication. A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis. A functional copy of IS
1629
from
S. turgidiscabies
94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the λ
cI
857 repressor. IS
1629
is present in multiple copies in some
S. scabies
strains and is present in all
S. acidiscabies
and
S. turgidiscabies
strains examined. A second copy of IS
1629
was identified between ORF
tnp
and
nec1
in
S. acidiscabies
strains. The diversity of IS
1629
hybridization profiles was greatest within
S. scabies
. IS
1629
was absent from the 27 nonpathogenic
Streptomyces
strains tested. The genetic organization and nucleotide sequence of the
nec1
-IS
1629
region was conserved and identical among representatives of
S. acidiscabies
and
S. turgidiscabies
. These findings support our current model for the unidirectional transfer of the ORF
tnp-nec1
-IS
1629
locus from IS
1629
-containing
S. scabies
(type II) to
S. acidiscabies
and
S. turgidiscabies
.
Unusual Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase of Anoxic
Archaea
Authors: Watson Gregory M. F. , Yu Jae-Pil , Tabita F. Robert 
Published: December 2019

ABSTRACT

The predominant pool of organic matter on earth is derived from the biological reduction and assimilation of carbon dioxide gas, catalyzed primarily by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). By virtue of its capacity to use molecular oxygen as an alternative and competing gaseous substrate, the catalytic efficiency of RubisCO and the enzyme’s ability to assimilate CO
2
may be severely limited, with consequent environmental and agricultural effects. Recent genomic sequencing projects, however, have identified putative RubisCO genes from anoxic
Archaea
. In the present study, these potential RubisCO sequences, from
Methanococcus jannaschii
and
Archaeoglobus fulgidus
, were analyzed in order to ascertain whether such sequences might encode functional proteins. We also report the isolation and properties of recombinant RubisCO using sequences obtained from the obligately anaerobic hyperthermophilic methanogen
M. jannaschii
. This is the first description of an archaeal RubisCO sequence; this study also represents the initial characterization of a RubisCO molecule that has evolved in the absence of molecular oxygen. The enzyme was shown to be a homodimer whose deduced sequence, along with other recently obtained archaeal RubisCO sequences, differs substantially from those of known RubisCO molecules. The recombinant
M. jannaschii
enzyme has a somewhat low, but reasonable
k
cat
, however, unlike previously isolated RubisCO molecules, this enzyme is very oxygen sensitive yet it is stable to hyperthermal temperatures and catalyzes the formation of the expected carboxylation product. Despite inhibition by oxygen, this unusual RubisCO still catalyzes a weak yet demonstrable oxygenase activity, with perhaps the lowest capacity for CO
2
/O
2
discrimination ever encountered for any RubisCO.
CspI, the Ninth Member of the CspA Family of
Escherichia coli
, Is Induced upon Cold Shock
Authors: Wang Nan , Yamanaka Kunitoshi , Inouye Masayori 
Published: December 2019

ABSTRACT

Escherichia coli
contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The
cspI
gene is located at 35.2 min on the
E. coli
chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of
cspI-lacZ
fusion constructs and the
cspI
mRNA revealed that
cspI
is cold shock inducible. The 5′-untranslated region of the
cspI
mRNA consists of 145 bases and causes a negative effect on
cspI
expression at 37°C. The
cspI
mRNA was very unstable at 37°C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15°C. Taking these results together,
E. coli
possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10°C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10°C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10°C).
Metal-Catalyzed Oxidation of Phenylalanine-Sensitive 3-Deoxy-
d
-
arabino
-Heptulosonate-7-Phosphate Synthase from
Escherichia coli
: Inactivation and Destabilization by Oxidation of Active-Site Cysteines
Authors: Park Ohkmae K. , Bauerle Ronald 
Published: December 2019

ABSTRACT

The in vitro instability of the phenylalanine-sensitive 3-deoxy-
d
-
arabino
-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from
Escherichia coli
has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and
d
-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu
2+
and Fe
2+
, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu
2+
-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu
2+
. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu
2+
-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys
61
or Cys
328
, were insensitive to the metal attack. Peptide mapping of the Cu
2+
-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.
Model for Bacteriophage T4 Development in
Escherichia coli
Authors: Rabinovitch Avinoam , Hadas Hilla , Einav Monica , Melamed Zeev , Zaritsky Arieh 
Published: December 2019

ABSTRACT

Mathematical relations for the number of mature T4 bacteriophages, both inside and after lysis of an
Escherichia coli
cell, as a function of time after infection by a single phage were obtained, with the following five parameters: delay time until the first T4 is completed inside the bacterium (eclipse period, ν) and its standard deviation (ς), the rate at which the number of ripe T4 increases inside the bacterium during the rise period (α), and the time when the bacterium bursts (μ) and its standard deviation (β). Burst size [
B
= α(μ − ν)], the number of phages released from an infected bacterium, is thus a dependent parameter. A least-squares program was used to derive the values of the parameters for a variety of experimental results obtained with wild-type T4 in
E. coli
B/r under different growth conditions and manipulations (H. Hadas, M. Einav, I. Fishov, and A. Zaritsky, Microbiology 143:179–185, 1997). A “destruction parameter” (ζ) was added to take care of the adverse effect of chloroform on phage survival. The overall agreement between the model and the experiment is quite good. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations.
Genomic Complexity among Strains of the Facultative Photoheterotrophic Bacterium
Rhodobacter sphaeroides
Authors: Nereng Kirsten Siedenburg , Kaplan Samuel 
Published: December 2019

ABSTRACT

Pulsed-field gel electrophoresis following the use of rare cutting restriction endonucleases together with Southern hybridization, using markers distributed on chromosomes I and II of
Rhodobacter sphaeroides
2.4.1, has been used to examine approximately 25 strains of
R. sphaeroides
in an effort to assess the occurrence of genome complexity in these strains. The results suggest that genome complexity is widespread and is accompanied by substantial genomic heterogeneity.
The Level of Expression of the Minor Pilin Subunit, CooD, Determines the Number of CS1 Pili Assembled on the Cell Surface of
Escherichia coli
Authors: Sakellaris Harry , Penumalli Vikram R. , Scott June R. 
Published: December 2019

ABSTRACT

CooD, the minor subunit of CS1 pili of enterotoxigenic
Escherichia coli
, is essential for the assembly of stable, functional pili. We previously proposed that CooD is a rate-limiting initiator of CS1 pilus assembly and predicted that the level of CooD expression should therefore determine the number of CS1 pili assembled on the cell surface. In this study, we confirm that CooD is required for the initiation of pilus assembly rather than for the stabilization of pili after they are assembled by demonstrating that specific modulation of
cooD
expression also modulates the number of CS1 pili on bacterial cells.
Characterization of the Dimerization Domain in BglG, an RNA-Binding Transcriptional Antiterminator from
Escherichia coli
Authors: Boss Amarelle , Nussbaum-Shochat Anat , Amster-Choder Orna 
Published: December 2019

ABSTRACT

The
Escherichia coli
transcriptional antiterminator protein BglG inhibits transcription termination of the
bgl
operon in response to the presence of β-glucosides in the growth medium. BglG is an RNA-binding protein that recognizes a specific sequence partially overlapping the two terminators within the
bgl
transcript. The activity of BglG is determined by its dimeric state which is modulated by reversible phosphorylation. Thus, only the nonphosphorylated dimer binds to the RNA target site and allows readthrough of transcription. Genetic systems which test dimerization and antitermination in vivo were used to map and delimit the region which mediates BglG dimerization. We show that the last 104 residues of BglG are required for dimerization. Any attempt to shorten this region from the ends or to introduce internal deletions abolished the dimerization capacity of this region. A putative leucine zipper motif is located at the N terminus of this region. The role of the canonical leucines in dimerization was demonstrated by their substitution. Our results also suggest that the carboxy-terminal 70 residues, which follow the leucine zipper, contain another dimerization domain which does not resemble any known dimerization motif. Each of these two regions is necessary but not sufficient for dimerization. The BglG phosphorylation site, His
208
, resides at the junction of the two putative dimerization domains. Possible mechanisms by which the phosphorylation of BglG controls its dimerization and thus its activity are discussed.
Organization of Biogenesis Genes for Aggregative Adherence Fimbria II Defines a Virulence Gene Cluster in Enteroaggregative
Escherichia coli
Authors: Elias Waldir P. , Czeczulin John R. , Henderson Ian R. , Trabulsi Luiz R. , Nataro James P. 
Published: December 2019

ABSTRACT

Several virulence-related genes have been described for prototype enteroaggregative
Escherichia coli
(EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.
Analysis of Elements Involved in Pseudoknot-Dependent Expression and Regulation of the
repA
Gene of an IncL/M Plasmid
Authors: Athanasopoulos V. , Praszkier J. , Pittard A. J. 
Published: December 2019

ABSTRACT

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the
repA
mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of
repA
, its presence was shown to interfere with the translation of
repB
. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of
repA
, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the
repA
SD was shown to be suboptimal for maximal expression of
repA
.
Identification of the
tliDEF
ABC Transporter Specific for Lipase in
Pseudomonas fluorescens
SIK W1
Authors: Ahn Jung Hoon , Pan Jae Gu , Rhee Joon Shick 
Published: December 2019

ABSTRACT

Pseudomonas fluorescens
, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of
P. fluorescens
SIK W1 was cloned and expressed in
Escherichia coli
, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter,
tliD
,
tliE
, and
tliF
, upstream of the lipase gene,
tliA
. In addition, genes encoding a protease and a protease inhibitor were located upstream of
tliDEF
.
tliDEF
showed high similarity to ABC transporters of
Pseudomonas aeruginosa
alkaline protease,
Erwinia chrysanthemi
protease,
Serratia marcescens
lipase, and
Pseudomonas fluorescens
CY091 protease.
tliDEF
and the lipase structural gene in a single operon were sufficient for
E. coli
cells to secrete the lipase. In addition,
E. coli
harboring the lipase gene secreted the lipase by complementation of
tliDEF
in a different plasmid. The ABC transporter of
P. fluorescens
was optimally functional at 20 and 25°C, while the ABC transporter,
aprD
,
aprE
, and
aprF
, of
P. aeruginosa
secreted the lipase irrespective of temperature between 20 and 37°C. These results demonstrated that the lipase is secreted by the
P. fluorescens
SIK W1 ABC transporter, which is organized as an operon with
tliA
, and that its secretory function is temperature dependent.