bluuebunny/crossref_metadata_2025_split huggingface dataset:
Promoter Selectivity of the
Bradyrhizobium japonicum
RpoH Transcription Factors In Vivo and In Vitro
Authors: Narberhaus Franz , Kowarik Michael , Beck Christoph , Hennecke Hauke 
Published: December 2019

ABSTRACT

Expression of the
dnaKJ
and
groESL
1
heat shock operons of
Bradyrhizobium japonicum
depends on a ς
32
-like transcription factor. Three such factors (RpoH
1
, RpoH
2
, and RpoH
3
) have previously been identified in this organism. We report here that they direct transcription from some but not all ς
32
-type promoters when the respective
rpoH
genes are expressed in
Escherichia coli
. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH
3
was insoluble. The purified proteins were recognized by an anti-
E. coli
ς
32
serum. While RpoH
1
and RpoH
2
productively interacted with
E. coli
core RNA polymerase and produced
E. coli groE
transcript in vitro, RpoH
3
was unable to do so.
B. japonicum
core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH
1
and RpoH
2
were active, and they initiated transcription at the
B. japonicum groESL
1
and
dnaKJ
promoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the
B. japonicum dnaKJ
and
groESL
1
promoters were suboptimal for transcription by RpoH
1
- or RpoH
2
-containing RNA polymerase from
B. japonicum
. In a mixture of different templates, the
E. coli groESL
promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward
B. japonicum rpoH
-dependent promoters. We conclude that the primary function of RpoH
2
is to supply the cell with DnaKJ under normal growth conditions whereas RpoH
1
is responsible mainly for increasing the level of GroESL
1
after a heat shock.
NADH Dehydrogenase Defects Confer Isoniazid Resistance and Conditional Lethality in
Mycobacterium smegmatis
Authors: Miesel Lynn , Weisbrod Torin R. , Marcinkeviciene Jovita A. , Bittman Robert , Jacobs William R. 
Published: December 2019

ABSTRACT

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of
Mycobacterium tuberculosis
infections. Resistance to INH in clinical isolates has been correlated with mutations in the
inhA
,
katG
, and
ahpC
genes. In this report, we describe a new mechanism for INH resistance in
Mycobacterium smegmatis
. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the
M. tuberculosis
complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD
+
ratio confers INH resistance.
Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex
Authors: We˛grzyn Alicja , Herman-Antosiewicz Anna , Taylor Karol , We˛grzyn Grzegorz 
Published: December 2019

ABSTRACT

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the
Escherichia coli
ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the
groE
operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λ
cro
+
, in λ
cro
−
plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λ
cro
ts plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λ
cro
−
plasmid replication control.
Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants
Authors: Nelson Bryn D. , Traxler Beth 
Published: December 2019

ABSTRACTThe maltose transport complex ofEscherichia coliis a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.
Identification of an
Escherichia coli pepA
Homolog and Its Involvement in Suppression of the
algB
Phenotype in Mucoid
Pseudomonas aeruginosa
Authors: Woolwine Samuel C. , Wozniak Daniel J. 
Published: December 2019

ABSTRACT

Strains of
Pseudomonas aeruginosa
isolated from the respiratory tracts of patients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regulator AlgB is required for full transcription of the alginate biosynthetic operon. Repeated attempts to demonstrate a direct interaction between AlgB and the promoter region of
algD
, the first gene in the alginate operon, have thus far been unsuccessful. The possibility that AlgB exerts its effect on
algD
indirectly exists. To identify putative genes under the control of AlgB which affect
algD
transcription, transposon mutagenesis of nonmucoid
algB
derivatives of the mucoid strain FRD1 was employed. Of approximately 3,000 transposon mutants screened, 6 were found to display phenotypes which were mucoid relative to the phenotype of the parental
algB
strain. The phenotypes of these mutants ranged from being only slightly mucoid to being indistinguishable from that of the original FRD1 strain. One of the particularly mucoid transposon mutants was chosen for further study. This strain was found to be disrupted in a previously uncharacterized open reading frame with 56% amino acid identity to PepA of
Escherichia coli
. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bacterial, plant, and animal species. This novel gene has been designated
phpA
(
P. aeruginosa
homolog of
pepA
). The insertional inactivation of
phpA
was found to correlate with the mucoid phenotype and an increase in
algD
transcription in the
algB
strain. Expression of
phpA
from an ectopic chromosomal locus compensated for the transposon insertion in the native
phpA
gene, restoring
algD
transcription to levels similar to those observed in the parental
algB
strain. While
phpA
expression did not appear to be under the control of AlgB at the transcriptional level, this study demonstrates that loss of
phpA
in an
algB
genetic background had a positive effect on alginate expression and, more specifically, on transcription of the alginate biosynthetic operon.
Chromosome Map of
Xanthomonas campestris
pv. campestris 17 with Locations of Genes Involved in Xanthan Gum Synthesis and Yellow Pigmentation
Authors: Tseng Yi-Hsiung , Choy Ka-Tim , Hung Chih-Hsin , Lin Nien-Tsung , Liu Jane-Yu , Lou Chih-Hong , Yang Bih-Ying , Wen Fu-Shyan , Weng Shu-Fen , Wu Jung-Rung 
Published: December 2019

ABSTRACT

No plasmid was detected in
Xanthomonas campestris
pv. campestris 17, a strain of the causative agent of black rot in cruciferous plants isolated in Taiwan. Its chromosome was cut by
Pac
I,
Pme
I, and
Swa
I into five, two, and six fragments, respectively, and a size of 4.8 Mb was estimated by summing the fragment lengths in these digests. Based on the data obtained from partial digestion and Southern hybridization using probes common to pairs of the overlapping fragments or prepared from linking fragments, a circular physical map bearing the
Pac
I,
Pme
I, and
Swa
I sites was constructed for the
X. campestris
pv. campestris 17 chromosome. Locations of eight
eps
loci involved in exopolysaccharide (xanthan gum) synthesis, two
rrn
operons each possessing an unique
I-Ceu
I site, one
pig
cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn
5
(pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Km
r
) genes, which upon insertion introduced additional sites into the chromosome. The
recA
and
tdh
genes, with known sequences, were mapped by tagging with the polylinker-Km
r
segment from Tn
5
(pfm)CmKm. This is the first map for
X. campestris
and would be useful for genetic studies of this and related
Xanthomonas
species.
Sequence Diversity of Flagellin (fliC) Alleles in PathogenicEscherichia coli
Authors: Reid Sean D. , Selander Robert K. , Whittam Thomas S. 
Published: December 2019

ABSTRACTTo study the molecular evolution of flagellin, the protein subunit specifying flagellar (H) antigens, thefliCgenes from 15 pathogenic strains ofEscherichia coliwere amplified by PCR and sequenced. Comparison offliCsequences of H6 and H7 strains revealed that alleles have a mosaic structure indicating the occurrence of past horizontal transfer of DNA segments between strains. The close similarity of H7 sequences also indicates the exchange of an entirefliCH7 allele between distant clonal lineages. In addition, the ratio of silent substitutions to amino acid replacements suggests that a short segment in the central region offliChas been under positive selection in the divergence of H6 and H7 alleles. Phylogenetic analysis demonstrates that thefliCsequences of O157:H7 and O55:H7 serotypes are nearly identical and highly divergent from those ofE. colistrains expressing H6 and H2 flagellar antigens. A nonmotile clone of sorbitol-fermenting O157 has rapidly accumulated multiple mutations infliC, presumably as a result of the silencing of flagellin expression.
Recruitment of ZipA to the Septal Ring of
Escherichia coli
Is Dependent on FtsZ and Independent of FtsA
Authors: Hale Cynthia A. , de Boer Piet A. J. 
Published: December 2019

ABSTRACT

Cell division in prokaryotes is mediated by the septal ring. In
Escherichia coli
, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.
An Endoglucanase, EglA, from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Hydrolyzes β-1,4 Bonds in Mixed-Linkage (1→3),(1→4)-β-
d
-Glucans and Cellulose
Authors: Bauer Michael W. , Driskill Lance E. , Callen Walter , Snead Marjory A. , Mathur Eric J. , Kelly Robert M. 
Published: December 2019

ABSTRACT

The
eglA
gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon
Pyrococcus furiosus
, was cloned and expressed in
Escherichia coli
. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The
P. furiosus
endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and
p
-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.
SSB
, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes
Authors: Lopez Nelson , Halladay John , Walter William , Craig Elizabeth A. 
Published: December 2019

ABSTRACT

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a
Saccharomyces cerevisiae
Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of
SSB
under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele,
EXA3-1
, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the
EXA3-1
mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of
SSB
, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of
SSB
and ribosomal mRNA after a temperature upshift.
Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo
Authors: Dekker Niek , Tommassen Jan , Verheij Hubertus M. 
Published: December 2019

ABSTRACT

Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin from
Escherichia coli
. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.
The Type II Pullulanase of
Thermococcus hydrothermalis
: Molecular Characterization of the Gene and Expression of the Catalytic Domain
Authors: Erra-Pujada Marta , Debeire Philippe , Duchiron Francis , O’Donohue Michael J. 
Published: December 2019

ABSTRACT

The gene encoding a hyperthermostable type II pullulanase produced by
Thermococcus hydrothermalis
(Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (
apuA
) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.
Regulation of β-Galactosidase Expression in
Bacillus megaterium
DSM319 by a XylS/AraC-Type Transcriptional Activator
Authors: Strey Jan , Wittchen Klaus D. , Meinhardt Friedhelm 
Published: December 2019

ABSTRACT

The β-galactosidase-encoding
bgaM
gene of
Bacillus megaterium
DSM319 and the divergently orientated
bgaR
operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of
bgaM
; a potential regulator binding site overlaps the
bgaM
promoter. A mechanism for regulation of β-galactosidase expression in
B. megaterium
is proposed.
SufS Is a NifS-Like Protein, and SufD Is Necessary for Stability of the [2Fe-2S] FhuF Protein in
Escherichia coli
Authors: Patzer Silke I. , Hantke Klaus 
Published: December 2019

ABSTRACT

Escherichia coli fhuF
mutants, a
sufS
::MudI mutant, and a
sufD
::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the
sufS
and
sufD
genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the
sufS
open reading frame corresponds to
orf f406
. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream
sufD
gene (
orf f423
) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a Δ
sufD
strain failed. Radioactive labeling of N-His-FhuF with [
35
S]methionine showed that the protein was unstable in the Δ
sufD
mutant.
A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-
p
-Dioxin Pathway Genes in
Sphingomonas
sp. Strain RW1
Authors: Armengaud Jean , Timmis Kenneth N. , Wittich Rolf-Michael 
Published: December 2019

ABSTRACT

The bacterium
Sphingomonas
sp. strain RW1 is able to use dibenzo-
p
-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp
Hin
dIII fragment downstream of cistrons
dxnA1A2
, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in
Escherichia coli
. The first three ORFs of the locus, designated
dxnC
, ORF2, and
fdx3
, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively.
dxnD
encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and
dxnE
codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene,
dxnF
, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-
cis,cis
-muconate. The heteromeric protein encoded by
dxnGH
is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas
dxnI
specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-
p
-dioxin in
Sphingomonas
sp. strain RW1 thus suggests itself.