bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Membrane-Associated Protein, FliX, Is Required for an Early Step in
Caulobacter
Flagellar Assembly
Authors: Mohr Christian D. , MacKichan Joanna K. , Shapiro Lucy 
Published: December 2019

ABSTRACT

The ordered assembly of the
Caulobacter crescentus
flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarachy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene,
fliX
. A
fliX
null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed
fliX
within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The
fliX
gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of
fliX
is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in
Caulobacter
predivisional cells. Full expression of
fliX
was found to be dependent on
ctrA
, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the
fliX
promoter. The
fliX
gene is located upstream and is divergently transcribed from the class III flagellar gene
flgI
, which encodes the basal body P-ring monomer. Analysis of the
fliX-flgI
intergenic region revealed an arrangement of
cis
-acting elements similar to that of another set of
Caulobacter
class II and class III flagellar genes,
fliL-flgF
, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.
The
Bacillus subtilis
DinR Binding Site: Redefinition of the Consensus Sequence
Authors: Winterling Kevin W. , Chafin David , Hayes Jeffery J. , Sun Ji , Levine Arthur S. , Yasbin Ronald E. , Woodgate Roger 
Published: December 2019

ABSTRACT

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium
Bacillus subtilis
. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N
4
-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the
dinA
,
dinB
,
dinC
, and
dinR
genes. Furthermore, detailed mutational analysis of the
B. subtilis recA
operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N
4
region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the
B. subtilis dinR
and
recA
DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.
Nuclease Overexpression Mutants of
Serratia marcescens
Authors: Guynn Linda J. , Dai Wenping , Benedik Michael J. 
Published: December 2019

ABSTRACT

A family of mutants overexpressing the
Serratia marcescens
extracellular nuclease has been known for decades. A number of these alleles are characterized here at the molecular level, and the mutant genes are identified, yielding a likely model for their phenotype. The known mutations exert their effect indirectly on
nucA
expression by elevating the basal SOS response of the cell. Mutations have been found in
xerC
and
uvrD
, both of which result in partial SOS induction. A classic

nuc
su

allele, that of strain W1050, is also likely to be in
xerC.
The Pol β-14 Dominant Negative Rat DNA Polymerase β Mutator Mutant Commits Errors during the Gap-Filling Step of Base Excision Repair in
Saccharomyces cerevisiae
Authors: Clairmont Caroline A. , Sweasy Joann B. 
Published: December 2019

ABSTRACT

We demonstrated recently that dominant negative mutants of rat DNA polymerase β (Pol β) interfere with repair of alkylation damage in
Saccharomyces cerevisiae
. To identify the alkylation repair pathway that is disrupted by the Pol β dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol β mutants to genes known to be involved in repair of DNA alkylation damage in
S. cerevisiae
. We demonstrate that the rat Pol β mutants interfere with the base excision repair pathway in
S. cerevisiae
. In addition, expression of one of the Pol β dominant negative mutants, Pol β-14, increases the spontaneous mutation rate of
S. cerevisiae
whereas expression of another Pol β dominant negative mutant, Pol β-TR, does not. Expression of the Pol β-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol β-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.
Activation of the Proprotein Transcription Factor Pro-ς
E
Is Associated with Its Progression through Three Patterns of Subcellular Localization during Sporulation in
Bacillus subtilis
Authors: Hofmeister Antje 
Published: December 2019

ABSTRACT

The activity of the sporulation transcription factor ς
E
in
Bacillus subtilis
is governed by an intercellular signal transduction pathway that controls the conversion of the inactive proprotein pro-ς
E
to the mature and active form of the factor. Here I use immunofluorescence microscopy to show that the activation of the proprotein is associated with its progression through three patterns of subcellular localization. In the predivisional sporangium, pro-ς
E
was found to be associated with the cytoplasmic membrane. Next, at the stage of asymmetric division, pro-ς
E
accumulated at the sporulation septum. Finally, after processing, mature ς
E
was found to be distributed throughout the mother cell cytoplasm. The results of subcellular fractionation and sedimentation in density gradients of extracts prepared from postdivisional sporangia confirmed that pro-ς
E
was chiefly present in the membrane fraction and that ς
E
was predominantly cytoplasmic, findings that suggest that the pro-amino acid sequence is responsible for the sequestration of pro-ς
E
to the membrane. The results of chemical cross-linking experiments showed that pro-ς
E
was present in a complex with its putative processing protein, SpoIIGA, or with a protein that depended on SpoIIGA. The membrane association of pro-ς
E
was, however, independent of SpoIIGA and other proteins specific to
B. subtilis
. Likewise, accumulation of pro-ς
E
at the septum did not depend on its interaction with SpoIIGA. Sequestration of pro-ς
E
to the membrane might serve to facilitate its interaction with SpoIIGA and may be important for preventing its premature association with core RNA polymerase. The implications of these findings for the compartmentalization of ς
E
are discussed.
Molecular Cloning of the Actinomycin Synthetase Gene Cluster from
Streptomyces chrysomallus
and Functional Heterologous Expression of the Gene Encoding Actinomycin Synthetase II
Authors: Schauwecker Florian , Pfennig Frank , Schröder Werner , Keller Ullrich 
Published: December 2019

ABSTRACT

The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (
acmA
) and ACMS II (
acmB
) by hybridization screening of a cosmid library of
Streptomyces chrysomallus
DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (
acmC
), is located immediately downstream of
acmB
in the same orientation. The protein sequence of ACMS II, deduced from
acmB
, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of
acmB
from the
mel
promoter of plasmid PIJ702 in
Streptomyces lividans
yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl–
l
-valine, which is epimerized to threonyl–
d
-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from
S. chrysomallus.
Thioredoxin Is Involved in Oxygen-Regulated Formation of the Photosynthetic Apparatus of
Rhodobacter sphaeroides
Authors: Pasternak Cecile , Haberzettl Kerstin , Klug Gabriele 
Published: December 2019

ABSTRACT

Thioredoxin, a redox active protein, has been previously demonstrated to be essential for growth of the anoxygenic photosynthetic bacterium
Rhodobacter sphaeroides
. In the present study, the involvement of thioredoxin in the formation of the photosynthetic apparatus of
R. sphaeroides
WS8 was investigated by construction and analysis of a mutant strain disrupted for the chromosomal
trxA
copy and carrying a plasmid-borne copy of
trxA
under the control of the hybrid p

trc

promoter inducible by IPTG (isopropyl-β-
d
-thiogalactopyranoside). This strain was viable in the absence of IPTG but was affected in pigmentation. When shifted from high to low oxygen tension conditions, the
trxA
mutant showed a reduced bacteriochlorophyll content in comparison to that of the wild type. Although thioredoxin is able to regulate aminolevulinic acid (ALA) synthase (the first enzyme of the tetrapyrrole biosynthetic pathway) activity by a dithiol-disulfide exchange, our mutant strain exhibited a level of ALA synthase activity identical to that of the wild type, suggesting that thioredoxin is involved in other steps to regulate the synthesis of the photosynthetic apparatus. Accordingly, we showed that the
trxA
mutation affects the oxygen-regulated expression of the
puf
operon encoding the pigment-binding proteins of the light-harvesting and reaction center complexes. Upon transition from aerobic to semiaerobic growth conditions, the maximal
puf
mRNA level was found to be 40 to 50% lower in the mutant strain than in the wild type. The stability of the
puf
transcripts was identical in both strains grown under low oxygen tension, indicating that the role of thioredoxin in regulating
puf
expression occurs at the transcriptional level.
Molecular and Biochemical Characterization of the Protein Template Controlling Biosynthesis of the Lipopeptide Lichenysin
Authors: Konz Dirk , Doekel Sascha , Marahiel Mohamed A. 
Published: December 2019

ABSTRACTLichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains ofBacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon fromB. licheniformisATCC 10716. Three large open reading frames coding for peptide synthetases, designatedlicA,licB(three modules each), andlicC(one module), could be detected, followed by a gene,licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed inEscherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequencel-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.
A Membrane-Bound NAD(P)
+
-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by
Klebsiella pneumoniae
Authors: Steuber Julia , Krebs Walter , Bott Michael , Dimroth Peter 
Published: December 2019

ABSTRACT

During anaerobic growth of
Klebsiella pneumoniae
on citrate, 9.4 mmol of H
2
/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO
2
. Upon addition of NiCl
2
(36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H
2
-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)
+
was also achieved by the addition of formate. In crude extracts, the H
2
:NAD
+
oxidoreductase activity was 0.13 μmol min
−1
mg
−1
, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min
−1
mg
−1
at pH 7.0 and 1.7 μmol of NADH formed min
−1
mg
−1
at pH 9.5. In the presence of the protonophore carbonyl cyanide
m-
chlorophenylhydrazone and the Na
+
/H
+
antiporter monensin, the H
2
-dependent reduction of NAD
+
by membrane vesicles decreased only slightly (about 16%). The NADP
+
- or NAD
+
-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-
N
-oxide or Triton X-100. NAD(P)H formation with H
2
as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by
K. pneumoniae
during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H
2
:NAD(P)
+
oxidoreductase that provides reducing equivalents for the synthesis of cell material.
Repair of Oxidized Bases in the Extremely Radiation-Resistant Bacterium
Deinococcus radiodurans
Authors: Bauche Cécile , Laval Jacques 
Published: December 2019

ABSTRACT

Deinococcus radiodurans
is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that
D. radiodurans
possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase–a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the
Escherichia coli
Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.
APT1
, but Not
APT2
, Codes for a Functional Adenine Phosphoribosyltransferase in
Saccharomyces cerevisiae
Authors: Alfonzo Juan D. , Crother Timothy R. , Guetsova Maria L. , Daignan-Fornier Bertrand , Taylor Milton W. 
Published: December 2019

ABSTRACT

The yeast
Saccharomyces cerevisiae
has two separate genes (
APT1
and
APT2
) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only
APT1
could code for a complementing activity. Cloning and expression of both the
APT1
and
APT2
genes in
Escherichia coli
showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only
APT1
was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that
APT1
is the functional gene in
S. cerevisiae
and that
APT2
is a pseudogene.
A Novel
Bacillus subtilis
Gene,
antE
, Temporally Regulated and Convergent to and Overlapping
dnaE
Authors: Wang Lin-Fa , Park Sung-Soo , Doi Roy H. 
Published: December 2019

ABSTRACT

A
Bacillus subtilis
promoter, Px, that functions in a convergent manner with the
sigA
operon promoter P3 has been found in the
sigA
operon. Promoter Px is turned on at the same time as promoter P3 during early sporulation. The transcript from promoter Px codes for a small protein with partial homology to the OmpR protein from
Escherichia coli
and also carries an untranslated sequence at its 3′ end that is complementary to the 5′ end of the P3 transcript, which codes for the ribosome binding site of
dnaE
. The gene controlled by Px has been called
antE
. The expression of
antE
does not require ς
B
, ς
E
, or ς
H
. Px was transcribed in vitro by the ς
A
holoenzyme and is the seventh promoter to be recognized in the ς
A
operon. A possible role for the
antE
gene during early sporulation is proposed.
Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA
Authors: Ueda Kumiko , Seki Tatsuji , Kudo Takuji , Yoshida Toshiomi , Kataoka Masakazu 
Published: December 2019

ABSTRACT

To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475
Streptomyces
strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity.
Growth of
Azospirillum irakense
KBC1 on the Aryl β-Glucoside Salicin Requires either
salA
or
salB
Authors: Faure Denis , Desair Jos , Keijers Veerle , Bekri My Ali , Proost Paul , Henrissat Bernard , Vanderleyden Jos 
Published: December 2019

ABSTRACT

The rhizosphere nitrogen-fixing bacterium
Azospirillum irakense
KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes,
salA
and
salB
, conferring β-glucosidase activity to
Escherichia coli
, have been identified in a cosmid library of
A. irakense
DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(
salA-salB
)
A. irakense
mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either
salA
or
salB
, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.
A HilA-Independent Pathway to
Salmonella typhimurium
Invasion Gene Transcription
Authors: Rakeman Jennifer L. , Bonifield Heather R. , Miller Samuel I. 
Published: December 2019

ABSTRACT

Salmonella typhimurium
invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within
Salmonella
pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci,
sirB
and
sirC
, which contribute to SPI1 gene transcription were defined.
sirC
is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype.
sirB
is required for maximal expression of
sirC
and consists of two open reading frames located near
kdsA
, a gene involved in lipopolysaccharide biosynthesis.
sirC
expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of
sirC
or
sirA
restores expression of a subset of SPI1 genes, including
invF
and
sspC
, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of
inv
expression is important for efficient assembly and function of the SPI1 TTSS.
Bacteriophage T4
rnh
(RNase H) Null Mutations: Effects on Spontaneous Mutation and Epistatic Interaction with
rII
Mutations
Authors: Bebenek Anna , Smith Leslie A. , Drake John W. 
Published: December 2019

ABSTRACT

The bacteriophage T4
rnh
gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4
rnh
null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Ac
r
) mutations. Because mutations in the related
Saccharomyces cerevisiae RAD27
gene display a remarkable duplication mutator phenotype, we further explored the impact of
rnh
mutations upon the mutation process. We observed that most Ac
r
mutants in an
rnh
+
strain contain
ac
mutations, whereas only roughly half of the Ac
r
mutants detected in an
rnh
Δ strain bear
ac
mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of
ac
mutations arising in
rnh
Δ and
rnh
+
backgrounds are indistinguishable. Thus, the increase in Ac
r
mutants in an
rnh
Δ background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of
rI
mutations in an
rnh
Δ background. In a screen that detects mutations at both the
rI
locus and the much larger
rII
locus, the r frequency was severalfold lower in an
rnh
Δ background. This decrease was due to the phenotype of
rnh rII
double mutants, which display an r
+
plaque morphology but retain the characteristic inability of
rII
mutants to grow on λ lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process.