bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the
dpr
Product in Oxygen Tolerance in
Streptococcus mutans
Authors: Yamamoto Yuji , Higuchi Masako , Poole Leslie B. , Kamio Yoshiyuki 
Published: July 2002

ABSTRACT

We have previously identified and characterized the alkyl hydroperoxide reductase of
Streptococcus mutans
, which consists of two components, Nox-1 and AhpC. Deletion of both
nox-1
and
ahpC
had no effect on the sensitivity of
S. mutans
to cumene hydroperoxide or H
2
O
2
, implying that the existence of another antioxidant system(s) independent of the Nox-1–AhpC system compensates for the deficiency. Here, a new antioxidant gene (
dpr
for Dps-like peroxide resistance gene) was isolated from the
S. mutans
chromosome by its ability to complement an
ahpCF
deletion mutant of
Escherichia coli
with a
tert
-butyl hydroperoxide-hypersensitive phenotype. The
dpr
gene complemented the defect in peroxidase activity caused by the deletion of
nox-1
and
ahpC
in
S. mutans
. Under aerobic conditions, the
dpr
disruption mutant carrying a spectinomycin resistance gene (
dpr
::Spc
r
mutant) grew as well as wild-type
S. mutans
in liquid medium. However, the
dpr
::Spc
r
mutant could not form colonies on an agar plate under air. In addition, neither the
dpr
::Spc
r
ahpC
::Em
r
::
nox-1
triple mutant nor the
dpr
::Spc
r
sod
::Em
r
double mutant was able to grow aerobically in liquid medium. The 20-kDa
dpr
gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of
S. mutans
cells to air. We propose a mechanism by which Dpr confers aerotolerance on
S. mutans.
Mobilization of Chimeric
oriT
Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity
Authors: Fekete Richard A. , Frost Laura S. 
Published: July 2002

ABSTRACT

Cleavage at the F plasmid
nic
site within the origin of transfer (
oriT
) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at
nic
in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1
oriT
regions or various combinations of
nic
, TraY, and TraM binding sites, in addition to the
traM
gene. The efficiency of cleavage at
nic
and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F
traM
mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at
nic
was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of
nic
, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within
oriT
. High-affinity binding of TraY in
cis
to
oriT
allowed detection of cleavage at
nic
but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to
oriT
are preferentially targeted to the transfer apparatus (transferosome).
Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in
Lactococcus lactis
Authors: Melchiorsen Claus Rix , Jokumsen Kirsten Væver , Villadsen John , Johnsen Mads G. , Israelsen Hans , Arnau José 
Published: July 2002

ABSTRACT

The enzyme pyruvate formate-lyase (PFL) from
Lactococcus lactis
was produced in
Escherichia coli
and purified to obtain anti-PFL antibodies that were shown to be specific for
L. lactis
PFL. It was demonstrated that activated
L. lactis
PFL was sensitive to oxygen, as in
E. coli
, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of
L. lactis
, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In
E. coli
, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in
L. lactis
, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in
E. coli
and
L. lactis
.
Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of
metE
and
metH
: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase
Authors: Fritsch Paula S. , Urbanowski Mark L. , Stauffer George V. 
Published: July 2002

ABSTRACT

Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of
metE
and
metH
. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either
metE
only (residues 260, 276, 302, 306, 309, and 322) or
metH
only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for
metE
activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α
2
, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of
metE
, while MetR interacts preferentially with the αCTD on the β-associated α subunit at
metH
. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.
The Overlapping
angB
and
angG
Genes Are Encoded within the
trans
-Acting Factor Region of the Virulence Plasmid in
Vibrio anguillarum
: Essential Role in Siderophore Biosynthesis
Authors: Welch Timothy J. , Chai Sunghee , Crosa Jorge H. 
Published: July 2002

ABSTRACT

Products encoded in the
trans
-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of
Vibrio anguillarum
. Here we identify
angB
, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by
angB
and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2,3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of various
angB
mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designated
angG
, also has an essential biosynthetic function. The
angG
gene directs the expression of three polypeptides when overexpressed in
Escherichia coli
, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the
angB
and
angG
genes.
A Thermostable Single-Strand DNase from
Methanococcus jannaschii
Related to the RecJ Recombination and Repair Exonuclease from
Escherichia coli
Authors: Rajman Luis A. , Lovett Susan T. 
Published: July 2002

ABSTRACT

The RecJ protein of
Escherichia coli
plays an important role in a number of DNA repair and recombination pathways. RecJ catalyzes processive degradation of single-stranded DNA in a 5′-to-3′ direction. Sequences highly related to those encoding RecJ can be found in most of the eubacterial genomes sequenced to date. From alignment of these sequences, seven conserved motifs are apparent. At least five of these motifs are shared among a large family of proteins in eubacteria, eukaryotes, and archaea, including the PPX1 polyphosphatase of yeast and
Drosophila
Prune. Archaeal genomes are particularly rich in such sequences, but it has not been clear whether any of the encoded proteins play a functional role similar to that of RecJ exonuclease. We have investigated three such proteins from
Methanococcus jannaschii
with the strongest overall sequence similarity to
E. coli
RecJ. Two of the genes, MJ0977 and MJ0831, partially complement a
recJ
mutant phenotype in
E. coli
. The expression of MJ0977 in
E. coli
resulted in high levels of a thermostable single-stranded DNase activity with properties similar to those of RecJ exonuclease. Despite overall weak sequence similarity between the MJ0977 product and RecJ, these nucleases are likely to have similar biological functions.
Regulation by Overlapping Promoters of the Rate of Synthesis and Deposition into Crystalline Inclusions of
Bacillus thuringiensis
δ-Endotoxins
Authors: Sedlak Mira , Walter Thomas , Aronson Arthur 
Published: July 2002

ABSTRACT

During sporulation,
Bacillus thuringiensis
produces intracellular, crystalline inclusions comprised of a mixture of protoxins active on insect larvae. A major class of these protoxin genes, designated
cry1
, is transcribed from two overlapping promoters (BtI and BtII) utilizing RNA polymerase containing sporulation sigma factors ς
E
and ς
K
, respectively. Fusions of these promoters to
lacZ
were constructed in order to analyze transcription patterns. Mutations within the −10 region of the BtII promoter (within the spacer region of the BtI promoter) which departed from the consensus −10 sequence for either ς
E
or ς
K
resulted in inactivation of transcription from BtII and a fivefold stimulation of transcription from BtI. In contrast, transcription from both promoters was inhibited with a change to the ς
E
consensus. One of the “promoter-up” mutations was fused to the
cry1Ac1
gene, and enhanced transcription was confirmed by Northern blotting. There was an increase in the accumulation of Cry1Ac antigen at early but not later times in sporulation in the mutant. This shift was due to the rapid turnover of much of the excessively accumulated protoxin at the early times as measured by pulse-chase labeling. As a result of the turnover and the inactivation of the BtII promoter, the mutant produced smaller inclusions which contained two- to threefold-less protoxin than inclusions from the wild type. Promoter overlap is a mechanism for modulating protoxin synthesis, thus ensuring the efficient packaging of these protoxins into inclusions.
Global Regulatory Mutations in
csrA
and
rpoS
Cause Severe Central Carbon Stress in
Escherichia coli
in the Presence of Acetate
Authors: Wei Bangdong , Shin Sooan , LaPorte David , Wolfe Alan J. , Romeo Tony 
Published: July 2002

ABSTRACT

The
csrA
gene encodes a small RNA-binding protein, which acts as a global regulator in
Escherichia coli
and other bacteria (T. Romeo, Mol. Microbiol. 29:1321–1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of
csrA
in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of
csrA rpoS
mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (
glg
) mutants, which were no longer inhibited by acetate. Several classes of
glg
mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that
csrA
positively regulates
acs
(acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the
glg
mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the
csrA rpoS
strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.
A Study of AroP-PheP Chimeric Proteins and Identification of a Residue Involved in Tryptophan Transport
Authors: Cosgriff Angela J. , Brasier Geoff , Pi Jing , Dogovski Con , Sarsero Joseph P. , Pittard A. J. 
Published: July 2002

ABSTRACT
In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.
Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes
Authors: Šmajs David , Weinstock George M. 
Published: July 2002

ABSTRACT

The 5.2-kb ColJs plasmid of a colicinogenic strain of
Shigella sonnei
(colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the
rep
,
mob
, and
cer
regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (
cja
), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (
cji
) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (
cjl
) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the
cjl
gene was found upstream from
cja
. Moreover, the promoter upstream from
cjl
was similar to promoters described upstream from several colicin activity genes. The
cji
gene was found to be located downstream from
cja
with a transcription polarity opposite to that of the
cjl
and
cja
genes. The
cja
,
cji
, and
cjl
genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.
Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in
Bacillus subtilis
Authors: Inaoka Takashi , Kasai Koji , Ochi Kozo 
Published: July 2002

ABSTRACT

To investigate the function of ribosomal proteins and translational factors in
Bacillus subtilis
, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like
Escherichia coli
mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (
ram
) mutants from an S12 mutant. These
ram
mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a
ram
phenotype in
B. subtilis
. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.
Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole
Authors: Epple Ulrike D. , Suriapranata Ivet , Eskelinen Eeva-Liisa , Thumm Michael 
Published: July 2002

ABSTRACT

Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail. Using a
Saccharomyces cerevisiae
mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein. Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown.
aut5
Δ cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I. Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p. In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis. Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy. In
pep4
Δ cells immunogold electron microscopy located Aut5-Ha at ∼50-nm-diameter intravacuolar vesicles. Characteristic missorting in
vps
class E and
fab1
Δ cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway. In agreement with localization of Aut5-Ha at intravacuolar vesicles in
pep4
Δ cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A.
CheR- and CheB-Dependent Chemosensory Adaptation System of
Rhodobacter sphaeroides
Authors: Martin Angela C. , Wadhams George H. , Shah Deepan S. H. , Porter Steven L. , Mantotta Jeevani C. , Craig Tim J. , Verdult Peter H. , Jones Helen , Armitage Judith P. 
Published: July 2002

ABSTRACT

Rhodobacter sphaeroides
has multiple homologues of most of the
Escherichia coli
chemotaxis genes, organized in three major operons and other, unlinked, loci. These include
cheA

1

and
cheR

1

(
che
Op
1
) and
cheA

2

,

cheR
2

, and

cheB
1

(
che
Op
2
). In-frame deletions of these
cheR
and
cheB
homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR
2
and CheB
1
were essential for normal chemotaxis, whereas CheR
1
was not.
cheR

2

and
cheB

1

, but not
cheR

1

, were also able to complement the equivalent
E. coli
mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in
R. sphaeroides
. In
E. coli,
CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any
R. sphaeroides
chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR
2
either interacts with the NWETF motif of
E. coli
methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that
R. sphaeroides
has an adaptation system that is different from that of
Bacillus subtilis
and
E. coli,
with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA
2
, but not CheA
1
, is able to phosphorylate CheB
1
, suggesting that signaling through CheA
1
cannot initiate feedback receptor adaptation via CheB
1
-P.
Complementation of Cold Shock Proteins by Translation Initiation Factor IF1 In Vivo
Authors: Weber Michael H. W. , Beckering Carsten L. , Marahiel Mohamed A. 
Published: July 2002

ABSTRACT

The cold shock response in both
Escherichia coli
and
Bacillus subtilis
is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For
B. subtilis
it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the
B. subtilis cspB cspC
double deletion mutant revealed that removal of these
csp
genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from
E. coli
in a
B. subtilis cspB cspC
double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.