bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ribosome Release in Regulation of
tna
Operon Expression in
Escherichia coli
Authors: Konan Kouacou Vincent , Yanofsky Charles
Published: December 2019

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

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

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

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

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

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

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

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

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

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

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

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

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.
In Vivo Role of Catalase-Peroxidase in
Synechocystis
sp. Strain PCC 6803
Authors: Tichy Martin , Vermaas Wim
Published: December 2019

The
katG
gene coding for the only catalase-peroxidase in the cyanobacterium
Synechocystis
sp. strain PCC 6803 was deleted in this organism. Although the rate of H
2
O
2
decomposition was about 30 times lower in the Δ
katG
mutant than in the wild type, the strain had a normal phenotype and its doubling time as well as its resistance to H
2
O
2
and methyl viologen were indistinguishable from those of the wild type. The residual H
2
O
2
-scavenging capacity was more than sufficient to deal with the rate of H
2
O
2
production by the cell, estimated to be less than 1% of the maximum rate of photosynthetic electron transport in vivo. We propose that catalase-peroxidase has a protective role against environmental H
2
O
2
generated by algae or bacteria in the ecosystem (for example, in mats). This protective role is most apparent at a high cell density of the cyanobacterium. The residual H
2
O
2
-scavenging activity in the Δ
katG
mutant was a light-dependent peroxidase activity. However, neither glutathione peroxidase nor ascorbate peroxidase accounted for a significant part of this H
2
O
2
-scavenging activity. When a small thiol such as dithiothreitol was added to the medium, the rate of H
2
O
2
decomposition in the Δ
katG
mutant increased more than 10-fold, indicating that a thiol-specific peroxidase, for which thioredoxin may be the physiological electron donor, is present. Oxidized thioredoxin is likely to be reduced again by photosynthetic electron transport. Therefore, under laboratory conditions, there are only two enzymatic mechanisms for H
2
O
2
decomposition present in
Synechocystis
sp. strain PCC 6803. One is catalyzed by a catalase-peroxidase, and the other is catalyzed by thiol-specific peroxidase.
Genetic Analysis of the
Serratia marcescens
N28b O4 Antigen Gene Cluster
Authors: Saigí Francesc , Climent Núria , Piqué Núria , Sanchez Cesar , Merino Susana , Rubirés Xavier , Aguilar Alicia , Tomás Juan M. , Regué Miguel
Published: December 2019

The
Serratia marcescens
N28b
wbbL
gene has been shown to complement the
rfb-50
mutation of
Escherichia coli
K-12 derivatives, and a
wbbL
mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigı́, N. Piqué, N. Climent, S. Merino, S. Albertı́, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581–7586, 1997). We analyzed a recombinant cosmid containing the
wbbL
gene by subcloning and determination of O-antigen production phenotype in
E. coli
DH5α by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with
S. marcescens
O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an
E. coli
K-12 strain with a deletion of the
wb
cluster was used, suggesting that the O4
wb
cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a
wecA
mutant
E. coli
strain, suggesting that O4-antigen production is
wecA
dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the
S. marcescens
N28b
wbbA
insertion mutant and
wzm-wzt
deletion mutant, we suggest that the O4
wb
cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS
4
was found downstream from the last gene in the cluster (
wbbA
), suggesting that an IS
4
-like element could have been involved in the acquisition of the O4
wb
cluster.
Regulation of Autophosphorylation of
Escherichia coli
Nitrogen Regulator II by the PII Signal Transduction Protein
Authors: Jiang Peng , Ninfa Alexander J.
Published: December 2019

The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in
Escherichia coli
. The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated. The phosphatase activity of NRII is activated by the PII signal transduction protein. We showed that PII was also an inhibitor of the kinase activity of NRII. The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated. The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII. We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleavage of ATP by NRII. The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities. Under these conditions, the kinase activity of NRII is dominant. At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity. However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII’s kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).
Regulation of Expression of the
Lactococcus lactis
Histidine Operon
Authors: Delorme C. , Ehrlich S. D. , Renault P.
Published: December 2019

In
Lactococcus lactis
, the
his
operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5′ end of the transcript, named the
his
leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the
his
operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the
his
operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the
his
leader region were found to differ in seven distantly related strains of
L. lactis
. Analysis of the variable positions supports the folding model of the central part of the
his
leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the
his
operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the
L. lactis
operon by histidine.
The Acid-Inducible
asr
Gene in
Escherichia coli
: Transcriptional Control by the
phoBR
Operon
Authors: Sužiede˙liene˙ Edita , Sužiede˙lis Ke˛stutis , Garbenčiute˙ Vaida , Normark Staffan
Published: December 2019

Escherichia coli
responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, ∼450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated
asr
(for acid shock RNA), mapped at 35.98 min on the
E. coli
chromosome. Analysis of the
asr
DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The
asr
gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded
asr
::
lacZ
fusion under a native
asr
promoter was reduced ∼15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal
asr
was abolished in the presence of a
phoB-phoR
(a two-component regulatory system, controlling the
pho
regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the
asr
gene in the Δ(
phoB-phoR
) mutant strain was restored by introduction of the plasmid with cloned
phoB-phoR
genes. Primer extension analysis of the
asr
transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The
asr
promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the
phoB-phoR
regulatory system to sense an external acidity and regulate transcription of the
asr
gene.
BadR, a New MarR Family Member, Regulates Anaerobic Benzoate Degradation by
Rhodopseudomonas palustris
in Concert with AadR, an Fnr Family Member
Authors: Egland Paul G. , Harwood Caroline S.
Published: December 2019

A cluster of genes for the anaerobic degradation of benzoate has been described for the phototrophic bacterium
Rhodopseudomonas palustris
. Here we provide an initial analysis of the regulation of anaerobic benzoate degradation by examining the contributions of two regulators: a new regulator, BadR, encoded by the benzoate degradation gene cluster, and a previously described regulator, AadR, whose gene lies outside the cluster. Strains with single mutations in either
badR
or
aadR
grew slowly on benzoate but were relatively unimpaired in growth on succinate and several intermediates of benzoate degradation. A
badR aadR
double mutant was completely defective in anaerobic growth on benzoate. Effects of the regulators on transcriptional activation were monitored with an
R. palustris
strain carrying a chromosomal fusion of ′
lacZ
to the
badE
gene of the
badDEFG
operon. This operon encodes benzoyl-coenzyme A (benzoyl-CoA) reductase, an unusual oxygen-sensitive enzyme that catalyzes the benzene ring reduction reaction that is the rate-limiting step in anaerobic benzoate degradation. Expression of
badE
::′
lacZ
was induced 100-fold when cells grown aerobically on succinate were shifted to anaerobic growth on succinate plus benzoate. The
aadR
gene was required for a 20-fold increase in expression that occurred in response to anaerobiosis, and
badR
was responsible for a further 5-fold increase in expression that occurred in response to benzoate. Further studies with the
badE
::′
lacZ
fusion strain grown with various kinds of aromatic acids indicated that BadR probably responds to benzoyl-CoA acting as an effector molecule. Sequence information indicates that BadR is a member of the MarR family of transcriptional regulators. These studies expand the range of functions regulated by MarR family members to include anaerobic aromatic acid degradation and provide an example of a MarR-type protein that acts as a positive regulator rather than as a negative regulator, as do most MarR family members. AadR resembles the
Escherichia coli
Fnr regulator in sequence and contains cysteine residues that are spaced appropriately to serve in the capacity of a redox-sensing protein.
Binding Site Recognition by Rns, a Virulence Regulator in the AraC Family
Authors: Munson George P. , Scott June R.
Published: December 2019

The expression of CS1 pili by enterotoxigenic strains of
Escherichia coli
is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of P
coo
(the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of P
coo
in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic
E. coli
and the global regulator
virB
of
Shigella flexneri
.
The Major Phase-Variable Outer Membrane Protein of
Escherichia coli
Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR
Authors: Henderson Ian R. , Owen Peter
Published: December 2019

Here we report the characterization of an
Escherichia coli
gene (
agn43
) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The
agn43
gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α
43
subunit (predicted
M
r
of 49,789) and a C-terminal domain (β
43
) with features typical of a bacterial integral outer membrane protein (predicted
M
r
of 51,642). Secondary structure analysis predicts that β
43
exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic
Neisseria
and
Haemophilus
spp. The correct processing of the polyprotein to α
43
and β
43
in OmpT, OmpP, and DegP protease-deficient
E. coli
strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α
43
. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of
agn43
. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus,
oxyR
mutants are locked on for Ag43 expression, whereas
dam
mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the
agn43
gene.
Genetic Localization and Molecular Characterization of Two Key Genes (
mitAB
) Required for Biosynthesis of the Antitumor Antibiotic Mitomycin C
Authors: Mao Yingqing , Varoglu Mustafa , Sherman David H.
Published: December 2019

Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA),
d
-glucosamine, and carbamoyl phosphate. A gene (
mitA
) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus,
mrd
, in
Streptomyces lavendulae
. Nucleotide sequence analysis showed that
mitA
encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from
Amycolatopsis mediterranei
, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the
S. lavendulae
chromosomal copy of
mitA
completely blocked the production of MC. The function of
mitA
was confirmed by complementation of an
S. lavendulae
strain containing a K191A mutation in MitA with AHBA. A second gene (
mitB
) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of
mitA
that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products.
Acid- and Base-Induced Proteins during Aerobic and Anaerobic Growth of
Escherichia coli
Revealed by Two-Dimensional Gel Electrophoresis
Authors: Blankenhorn Darcy , Phillips Judith , Slonczewski Joan L.
Published: December 2019

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in
Escherichia coli
. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The
gad
system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.
Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph
Methylosulfonomonas methylovora
Authors: de Marco Paolo , Moradas-Ferreira Pedro , Higgins Timothy P. , McDonald Ian , Kenna Elizabeth M. , Murrell J. Colin
Published: December 2019

Methylosulfonomonas methylovora
M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp
Sph
I fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium,
Marinosulfonomonas methylotropha
, revealed the presence of
msm
genes highly related to those found in
Methylosulfonomonas
, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria.
Induction of Prophages of Enterohemorrhagic
Escherichia coli
O157:H7 with Norfloxacin
Authors: Matsushiro Aizo , Sato Koki , Miyamoto Hiroshi , Yamamura Tadashi , Honda Takeshi
Published: December 2019

Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic
Escherichia coli
O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of
stx
genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a
recA
mutant strain.
Structure-Function Study of MalF Protein by Random Mutagenesis
Authors: Tapia María Isabel , Mourez Michaël , Hofnung Maurice , Dassa Elie
Published: December 2019

MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of
malF
mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK
2
complex and/or in the transport mechanism.
The Choline-Converting Pathway in
Staphylococcus xylosus
C2A: Genetic and Physiological Characterization
Authors: Rosenstein Ralf , Futter-Bryniok Detlinde , Götz Friedrich
Published: December 2019

A
Staphylococcus xylosus
C2A gene cluster, which encodes enzymes in the pathway for choline uptake and dehydrogenation (
cud
), to form the osmoprotectant glycine betaine, was identified. The
cud
locus comprises four genes, three of which encode proteins with significant similarities to those known to be involved in choline transport and conversion in other organisms. The physiological role of the gene products was confirmed by analysis of
cud
deletion mutants. The fourth gene possibly codes for a regulator protein. Part of the gene cluster was shown to be transcriptionally regulated by choline and elevated NaCl concentrations as inducers.
Structure of a New Glycolipid from the
Mycobacterium avium-Mycobacterium intracellulare
Complex
Authors: Watanabe Motoko , Ohta Akihiro , Sasaki Shun-ichi , Minnikin David E.
Published: December 2019

From the lipid fraction of a freeze-dried cell mass of a strain of the
Mycobacterium avium-Mycobacterium intracellulare
complex, a new glycolipid was isolated and was characterized as 5-mycoloyl-α-arabinofuranosyl (1→1′)-glycerol, mainly on the basis of nuclear magnetic resonance spectroscopy studies.
NahW, a Novel, Inducible Salicylate Hydroxylase Involved in Mineralization of Naphthalene by
Pseudomonas stutzeri
AN10
Authors: Bosch Rafael , Moore Edward R. B. , García-Valdés Elena , Pieper Dietmar H.
Published: December 2019

Two genes,
nahG
and
nahW
, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain
Pseudomonas stutzeri
AN10. While
nahG
resides in the same transcriptional unit as the
meta
-cleavage pathway genes, forming the naphthalene degradation lower pathway,
nahW
is situated outside but in close proximity to this transcriptional unit. The
nahG
and
nahW
genes of
P. stutzeri
AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism. Both genes,
nahG
and
nahW
, have been cloned in
Escherichia coli
JM109. The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively. Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates. However, the relative rates by which the substituted analogs are transformed differ considerably.