bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation of the
Streptococcus mutans gal
Operon by the GalR Repressor
Authors: Ajdić Dragana , Ferretti Joseph J. 
Published: December 2019

ABSTRACT

The galactose operon of
Streptococcus mutans
is transcriptionally regulated by a repressor protein (GalR) encoded by the
galR
gene, which is divergently oriented from the structural genes of the
gal
operon. To study the regulatory function of GalR, we partially purified the protein and examined its DNA binding activity by gel mobility shift and DNase I footprinting experiments. The protein specifically bound to the
galR-galK
intergenic region at an operator sequence, the position of which would suggest that GalR plays a role in the regulation of the
gal
operon as well as autoregulation. To further examine this hypothesis, transcriptional start sites of the
gal
operon and the
galR
gene were determined. Primer extension analysis showed that both promoters overlap the operator, indicating that GalR most likely represses transcription initiation of both promoters. Finally, the results from in vitro binding experiments with potential effector molecules suggest that galactose is a true intracellular inducer of the galactose operon.
Subcellular Localization of
Bacillus subtilis
SMC, a Protein Involved in Chromosome Condensation and Segregation
Authors: Graumann Peter L. , Losick Richard , Strunnikov Alexander V. 
Published: December 2019

ABSTRACT

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium
Bacillus subtilis
. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the
B. subtilis
cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated
smc
gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.
Roles of Multiple Promoters in Transcription of Ribosomal DNA: Effects of Growth Conditions on Precursor rRNA Synthesis in Mycobacteria
Authors: Gonzalez-y-Merchand J. A. , Colston M. J. , Cox R. A. 
Published: December 2019

ABSTRACT

The roles of multiple promoters in the synthesis of rRNA under different conditions of growth were investigated, using two mycobacterial species as model organisms. When
Mycobacterium smegmatis
was grown under optimal conditions, its two rRNA operons contributed equally, with two promoters, one from each operon, being responsible for most transcripts. In stationary-phase growth or balanced growth under carbon starvation conditions, one operon (
rrnA
f
) dominated and its three promoters contributed more equally to the generation of transcripts.
Mycobacterium tuberculosis
has a single operon with two promoters, one of which generated 80% of transcripts, at all stages of growth. We infer that each promoter functions independently according to its intrinsic strength when cells are growing slowly so that one operon with three promoters is roughly equivalent to three operons with one promoter; at high growth rates, occlusion effects reduce the efficiency of multiple promoters to that of a single promoter.
Expression of Alkane Hydroxylase from
Acinetobacter
sp. Strain ADP1 Is Induced by a Broad Range of n- Alkanes and Requires the Transcriptional Activator AlkR
Authors: Ratajczak Andreas , Geißdörfer Walter , Hillen Wolfgang 
Published: December 2019

ABSTRACT

In
Acinetobacter
sp. strain ADP1, alkane degradation depends on at least five essential genes.
rubAB
and
xcpR
are constitutively transcribed. Here we describe inducible transcription of
alkM
, which strictly depends on the presence of the transcriptional activator AlkR.
alkR
itself is expressed at a low level, while a chromosomally located
alkM
::
lacZ
fusion is inducible by middle-chain-length alkanes from heptane to undecane, which do not support growth of ADP1, and by long-chain-length alkanes from dodecane to octadecane, which are used as sources of carbon and energy. The putative AlkM substrate 1-dodecene is also an effective inducer. Products of alkane hydroxylase activity like 1-dodecanol prevent induction of
alkM
expression.
alkM
is expressed only in stationary phase, suggesting its dependence on at least one other regulatory mechanism.
Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls
Authors: Hurtubise Yves , Barriault Diane , Sylvestre Michel 
Published: December 2019

ABSTRACT

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in
Comamonas testosteroni
B-356 and in
Pseudomonas
sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP
BPH
), a ferredoxin (FER
BPH
), and a ferredoxin reductase (RED
BPH
). B-356 BPH dox preferentially catalyzes the oxidation of the double-
meta
-substituted congener 3,3′-dichlorobiphenyl over the double-
para
-substituted congener 4,4′-dichlorobiphenyl or the double-
ortho
-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent
meta-para
carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the
meta-para
dioxygenation of chimeric enzymes obtained by exchanging the ISP
BPH
α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP
BPH
of strain LB400 and the β subunit of ISP
BPH
of strain B-356 (the α
LB400
β
B-356
chimera) and the α
B-356
β
LB400
chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP
BPH
β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α
B-356
β
LB400
chimera should have behaved like B-356 ISP
BPH
; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP
BPH
. On the other hand, the α
LB400
β
B-356
chimera showed features of both B-356 and LB400 ISP
BPH
where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the
meta-para
oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.
atp
Mutants of
Escherichia coli
Fail To Grow on Succinate Due to a Transport Deficiency
Authors: Boogerd Fred C. , Boe Lars , Michelsen Ole , Jensen Peter Ruhdal 
Published: December 2019

ABSTRACT

Escherichia coli atp
mutants, which lack a functional H
+
-ATPase complex, are capable of growth on glucose but not on succinate or other C
4
-dicarboxylates (Suc
−
phenotype). Suc
+
revertants of an
atp
deletion strain were isolated which were capable of growth on succinate even though they lack the entire H
+
-ATPase complex. Complementation in
trans
with the
yhiF
gene suppressed the growth of the Suc
+
mutants on succinate, which implicates the
yhiF
gene product in the regulation of C
4
-dicarboxylate metabolism. Indeed, when the
E. coli
C
4
-dicarboxylate transporter (encoded by the
dctA
gene) was expressed in
trans
, the Suc
−
phenotype of the
atp
deletion strain reverted to Suc
+
, which shows that the reason why the
E. coli atp
mutant is unable to grow aerobically on C
4
-dicarboxylates is insufficient transport capacity for these substrates.
A
Saccharomyces cerevisiae
Mutant Lacking a K
+
/H
+
Exchanger
Authors: Ramírez Jorge , Ramírez Oscar , Saldaña Carlos , Coria Roberto , Peña Antonio 
Published: December 2019

ABSTRACT

The
KHA1
gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K
+
/H
+
antiporter (873 amino acids) in
Saccharomyces cerevisiae
was disrupted by homologous recombination. The core protein is similar to the putative Na
+
/H
+
antiporters from
Enterococcus hirae
(
NAPA
gene) and
Lactococcus lactis
(
LLUPP
gene) and the putative K
+
/H
+
exchanger from
Escherichia coli
(
KEFC
gene). Disruption of the
KHA1
gene resulted in an increased K
+
accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of
86
Rb
+
uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K
+
accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.
Accumulation of the Enterobacterial Common Antigen Lipid II Biosynthetic Intermediate Stimulates
degP
Transcription in
Escherichia coli
Authors: Danese Paul N. , Oliver George R. , Barr Kathleen , Bowman Gregory D. , Rick Paul D. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, transcription of the
degP
locus, which encodes a heat-shock-inducible periplasmic protease, is controlled by two parallel signal transduction systems that each monitor extracytoplasmic protein physiology. For example, the heat-shock-inducible sigma factor, ς
E
, controls
degP
transcription in response to the overproduction and folded state of various extracytoplasmic proteins. Similarly, the CpxA/R two-component signal transduction system increases
degP
transcription in response to the overproduction of a variety of extracytoplasmic proteins. Since
degP
transcription is attuned to the physiology of extracytoplasmic proteins, we were interested in identifying negative transcriptional regulators of
degP
. To this end, we screened for null mutations that increased transcription from a strain containing a
degP-lacZ
reporter fusion. Through this approach, we identified null mutations in the
wecE
,
rmlA
ECA
, and
wecF
loci that increase
degP
transcription. Interestingly, each of these loci is responsible for synthesis of the enterobacterial common antigen (ECA), a glycolipid situated on the outer leaflet of the outer membrane of members of the family
Enterobacteriaceae
. However, these null mutations do not stimulate
degP
transcription by eliminating ECA biosynthesis. Rather, the
wecE
,
rmlA
ECA
, and
wecF
null mutations each impede the same step in ECA biosynthesis, and it is the accumulation of the ECA biosynthetic intermediate, lipid II, that causes the observed perturbations. For example, the lipid II-accumulating mutant strains each (i) confer upon
E. coli
a sensitivity to bile salts, (ii) confer a sensitivity to the synthesis of the outer membrane protein LamB, and (iii) stimulate both the Cpx pathway and ς
E
activity. These phenotypes suggest that the accumulation of lipid II perturbs the structure of the bacterial outer membrane. Furthermore, these results underscore the notion that although the Cpx and ς
E
systems function in parallel to regulate
degP
transcription, they can be simultaneously activated by the same perturbation.
Adherence of the Gram-Positive Bacterium
Ruminococcus albus
to Cellulose and Identification of a Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family of Proteins
Authors: Pegden Randall S. , Larson Marilynn A. , Grant Richard J. , Morrison Mark 
Published: December 2019

ABSTRACT

The adherence of
Ruminococcus albus
8 to crystalline cellulose was studied, and an affinity-based assay was also used to identify candidate cellulose-binding protein(s). Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both phenylacetic and phenylpropionic acids in the growth medium, and the addition of carboxymethylcellulose (CMC) to assays decreased the adherence of the bacterium to cellulose. A cellulose-binding protein with an estimated molecular mass following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ∼21 kDa, designated CbpC, was present in both cellobiose- and cellulose-grown cultures, and the relative abundance of this protein increased in response to growth on cellulose. Addition of 0.1% (wt/vol) CMC to the binding assays had an inhibitory effect on CbpC binding to cellulose, consistent with the notion that CbpC plays a role in bacterial attachment to cellulose. The nucleotide sequence of the
cbpC
gene was determined by a combination of reverse genetics and genomic walking procedures. The
cbpC
gene encodes a protein of 169 amino acids with a calculated molecular mass of 17,655 Da. The amino-terminal third of the CbpC protein possesses the motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in gram-negative, pathogenic bacteria. The remainder of the predicted CbpC sequence was found to have significant identity with 72- and 75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen protein of
Rickettsia
spp., as well as one of the major capsid glycoproteins of the
Chlorella
virus PBCV-1. Northern blot analysis showed that phenylpropionic acid and ruminal fluid increase
cbpC
mRNA abundance in cellobiose-grown cells. These results suggest that CbpC is a novel cellulose-binding protein that may be involved in adherence of
R. albus
to substrate and extends understanding of the distribution of the Pil family of proteins in gram-positive bacteria.
Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in
Methanothermus fervidus
Authors: Matussek Karl , Moritz Patrick , Brunner Nina , Eckerskorn Christoph , Hensel Reinhard 
Published: December 2019

ABSTRACT

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon
Methanothermus fervidus
, sequenced the coding gene, and expressed it in
Escherichia coli
. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in
M. fervidus
, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the
V
max
of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from
E. coli
revealed a twofold-higher
V
max
of the enzyme from
M. fervidus
in the synthesizing direction.
Role of the
parCBA
Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance
Authors: Easter Carla L. , Schwab Helmut , Helinski Donald R. 
Published: December 2019

ABSTRACT

The
par
region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,
parCBA
and
parDE
, and a
cis
-acting site.
parDE
encodes a postsegregational killing system, and
parCBA
encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the
parCBA
region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of
Escherichia coli
and two strains of
Pseudomonas aeruginosa
. The intact
parCBA
operon was effective in stabilizing a conjugation-defective RK2 derivative in
E. coli
MC1061K and RR1 but was relatively ineffective in
E. coli
MV10Δlac. In the two strains in which the
parCBA
operon was effective, deletions in
parB
,
parC
, or both
parB
and
parC
caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the
parA
gene resulted in a much greater loss of
parCBA
activity. For
P. aeruginosa
PAO1161Rif
r
, the
parCBA
operon provided little if any plasmid stability, but for
P. aeruginosa
PAC452Rif
r
, the RK2 plasmid was stabilized to a substantial extent by
parCBA
. With this latter strain,
parA
and
res
alone were sufficient for stabilization. The
cer
resolvase system of plasmid ColE1 and the
loxP
/Cre system of plasmid P1 were tested in comparison with the
parCBA
operon. We found that, not unlike what was previously observed with MC1061K,
cer
failed to stabilize the RK2 plasmid with
par
deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The
loxP
/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three
E. coli
strains. These observations indicate that the
parA
gene, along with its
res
site, exhibits a significant level of plasmid stabilization in the absence of the
parC
and
parB
genes but that in at least one
E. coli
strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with
parCBA
or the
cer
and
loxP
/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.
Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes inPseudomonassp. Strain 61-3
Authors: Matsusaki Hiromi , Manji Sumihide , Taguchi Kazunori , Kato Mikiya , Fukui Toshiaki , Doi Yoshiharu 
Published: December 2019

ABSTRACTTwo types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phbandpha) ofPseudomonassp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In thephblocus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), β-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed byphbBPs-phbAPs-phbCPs. Upstream fromphbBPswas found thephbRPsgene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. ThephbRPsgene was found to be transcribed in the opposite direction from the three structural genes. Cloning ofphbRPsin a relatively high-copy vector inPseudomonassp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptionalphbpromoter-lacZfusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPsis a positive regulatory protein controlling the transcription ofphbBACPsin this bacterium. In thephalocus, two genes encoding PHA synthases (PhaC1Psand PhaC2Ps) were flanked by a PHA depolymerase gene (phaZPs), and two adjacent open reading frames (ORF1 andphaDPs), and the gene order was ORF1,phaC1Ps,phaZPs,phaC2Ps, andphaDPs. Heterologous expression of the cloned fragments in PHA-negative mutants ofPseudomonas putidaandRalstonia eutropharevealed that PHB synthase and two PHA synthases ofPseudomonassp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.
IncP Plasmids Are Unusually Effective in Mediating Conjugation of
Escherichia coli
and
Saccharomyces cerevisiae
: Involvement of the Tra2 Mating System
Authors: Bates Steven , Cashmore Annette M. , Wilkins Brian M. 
Published: December 2019

ABSTRACT

Mobilizable shuttle plasmids containing the origin-of-transfer (
oriT
) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from
Escherichia coli
to
Saccharomyces cerevisiae
. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10
−5
transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of
oriT
in
cis
and the provision in
trans
of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (
trbB
, -
C
, -
D
, -
E
, -
F
, -
G
, -
H
, -
I
, -
J
, and -
L
) required for plasmid transfer to
E. coli
K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus
traF
is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.
Involvement of Both Dockerin Subdomains in Assembly of the
Clostridium thermocellum
Cellulosome
Authors: Lytle Betsy , Wu J. H. David 
Published: December 2019

ABSTRACT

Clostridium thermocellum
produces an extracellular cellulase complex termed the cellulosome. It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain. The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure. Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif. Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit. These subdomains, called DS1 and DS2, were cloned by PCR and expressed in
Escherichia coli
. The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis. A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.