bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Role of Catalase-Peroxidase in
Synechocystis
sp. Strain PCC 6803
Authors: Tichy Martin , Vermaas Wim 
Published: December 2019

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.