bluuebunny/crossref_metadata_2025_split huggingface dataset:
Visualization of Penicillin-Binding Proteins during Sporulation of
Streptomyces griseus
Authors: Hao Jiang , Kendrick Kathleen E. 
Published: December 2019

ABSTRACT

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of
Streptomyces griseus
. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of
S. griseus.
A Novel DNA Polymerase Family Found inArchaea
Authors: Ishino Yoshizumi , Komori Kayoko , Cann Isaac K. O. , Koga Yosuke 
Published: December 2019

ABSTRACTOne of the most puzzling results from the complete genome sequence of the methanogenic archaeonMethanococcus jannaschiiwas that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes inM. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosushave been located and cloned. The gene products ofM. jannaschiiexpressed inEscherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.
The
tfdK
Gene Product Facilitates Uptake of 2,4-Dichlorophenoxyacetate by
Ralstonia eutropha
JMP134(pJP4)
Authors: Leveau Johan H. J. , Zehnder Alexander J. B. , van der Meer Jan Roelof 
Published: December 2019

ABSTRACT

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by
Ralstonia eutropha
JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated
tfdK
, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the
tfdK
gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A
tfdA
mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.
Erwinia amylovora
Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, through the Hrp (Type III Secretion) Pathway
Authors: Bogdanove Adam J. , Bauer David W. , Beer Steven V. 
Published: December 2019

ABSTRACT

Erwinia amylovora
was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of
Pseudomonas syringae
pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an
hrp
gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.
S-Layered
Aneurinibacillus
and
Bacillus
spp. Are Susceptible to the Lytic Action of
Pseudomonas aeruginosa
Membrane Vesicles
Authors: Kadurugamuwa J. L. , Mayer A. , Messner P. , Sára M. , Sleytr U. B. , Beveridge T. J. 
Published: December 2019

ABSTRACT

When S-layered strains of
Bacillus stearothermophilus
and
Aneurinibacillus thermoaerophilus
, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from
P. aeruginosa
during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact.
meso
-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.
Control of 5′,5′-Dinucleoside Triphosphate Catabolism by
APH1
, a
Saccharomyces cerevisiae
Analog of Human
FHIT
Authors: Chen Josiane , Brevet Annie , Blanquet Sylvain , Plateau Pierre 
Published: December 2019

ABSTRACT

The putative human tumor suppressor gene
FHIT
(fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P
1
,P
3
-triphosphate (Ap
3
A) hydrolase. In this report, we show that the
Saccharomyces cerevisiae APH1
gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap
3
A being the preferred substrate. Accordingly, disruption of the
APH1
gene produced viable
S. cerevisiae
cells containing reduced Ap
3
A-hydrolyzing activity and a 30-fold-elevated Ap
3
N concentration.
Molecular Evolution of
Mycoplasma capricolum
subsp.
capripneumoniae
Strains, Based on Polymorphisms in the 16S rRNA Genes
Authors: Pettersson Bertil , Bölske Göran , Thiaucourt François , Uhlén Mathias , Johansson Karl-Erik 
Published: December 2019

ABSTRACT

Mycoplasma capricolum
subsp.
capripneumoniae
belongs to the so-called
Mycoplasma mycoides
cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the
M. mycoides
cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of
M. capricolum
subsp.
capripneumoniae
of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the
M. capricolum
subsp.
capripneumoniae
strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of
M. capricolum
subsp.
capripneumoniae
may be used as epidemiological markers for CCPP.
Gas Vesicle Genes Identified in
Bacillus megaterium
and Functional Expression in
Escherichia coli
Authors: Li Ning , Cannon Maura C. 
Published: December 2019

ABSTRACT

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (
gvp
) from
Bacillus megaterium
VT1660 and their functional expression in
Escherichia coli
. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of
E. coli
strains that carry this
gvp
gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-constrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In
B. megaterium
, the
gvp
region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and
gvpA
, -
P
, -
Q
, -
B
, -
R
, -
N
, -
F
, -
G
, -
L
, -
S
, -
K
, -
J
, -
T
, and -
U
, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in
E. coli
. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.
Division Planes Alternate in Spherical Cells of
Escherichia coli
Authors: Begg K. J. , Donachie W. D. 
Published: December 2019

ABSTRACT

In the spherical cells of
Escherichia coli rodA
mutants, division is initiated at a single point, from which a furrow extends progressively around the cell. Using “giant”
rodA ftsA
cells, we confirmed that each new division furrow is initiated at the midpoint of the previous division plane and runs perpendicular to it.
Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of
Streptomyces granaticolor
Authors: Nádvorník Richard , Vomastek Tomáš , Janeček Jiří , Techniková Zuzana , Branny Pavel 
Published: December 2019

ABSTRACT

A 4.2-kb
Sph
I-
Bam
HI fragment of chromosomal DNA from
Streptomyces granaticolor
was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The
pkg2
gene was overexpressed in
Escherichia coli
, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the
pkg2
gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
Mutations Affecting the Ability of the
Escherichia coli
UmuD′ Protein To Participate in SOS Mutagenesis
Authors: Ohta Toshihiro , Sutton Mark D. , Guzzo Angelina , Cole Shannon , Ferentz Ann E. , Walker Graham C. 
Published: December 2019

ABSTRACT

The products of the SOS-regulated
umuDC
operon are required for most UV and chemical mutagenesis in
Escherichia coli
, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable
umuD
mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190–7194, 1990). To identify elements of the UmuD′ protein necessary for its role in translesion synthesis, we began with
umuD
′, a modified form of the
umuD
gene that directly encodes the UmuD′ protein, and obtained missense
umuD
′ mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD′
2
homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded β-sheet and appears to interfere with UmuD′
2
homodimer formation indirectly by affecting the structure of the UmuD′ monomer. When expressed from a multicopy plasmid, the L40R
umuD
′ mutant gene exhibited a dominant negative effect on a chromosomal
umuD
+
gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD′ function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal β-strand and results in a mutant UmuD′ protein that exhibits a strongly dominant negative effect on UV mutagenesis in a
umuD
+
strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD′
2
homodimer, which are mobile in solution.
pSa Causes Oncogenic Suppression of
Agrobacterium
by Inhibiting VirE2 Protein Export
Authors: Lee Lan-Ying , Gelvin Stanton B. , Kado Clarence I. 
Published: December 2019

ABSTRACT

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the
osa
gene of the plasmid pSa can suppress crown gall tumorigenesis incited by
Agrobacterium tumefaciens
. Neither T-DNA processing nor
vir
(virulence) gene induction is affected by the presence of
osa
in the bacterium. We used
Arabidopsis thaliana
root segments and tobacco leaf discs to demonstrate that Osa inhibits
A. tumefaciens
from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable β-glucuronidase (GUS) expression. When
A. tumefaciens
contained
osa
, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of
Nicotiana benthamiana
by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an
A. tumefaciens virE2
mutant (the T-DNA donor strain) by an
A. tumefaciens
strain lacking T-DNA but containing a wild-type
virE2
gene (the VirE2 donor strain) was blocked when
osa
was present in the VirE2 donor strain, but not when
osa
was present in the T-DNA donor strain. These data indicate that
osa
inhibits VirE2 protein, but not T-DNA export from
A. tumefaciens
. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of
Datura stramonium
plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an
A. tumefaciens virE2
mutant carrying
osa
confirmed that oncogenic suppression by
osa
does not occur by blocking T-DNA transfer. Overexpression of
virB9
,
virB10
, and
virB11
in
A. tumefaciens
did not overcome oncogenic suppression by
osa
. The finding that the expression of the
osa
gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by
osa
may differ from that of the IncQ plasmid RSF1010.
Evidence that the Extracytoplasmic Function Sigma Factor ς
E
Is Required for Normal Cell Wall Structure in
Streptomyces coelicolor
A3(2)
Authors: Paget Mark S. B. , Chamberlin Leony , Atrih Abdelmadjid , Foster Simon J. , Buttner Mark J. 
Published: December 2019

ABSTRACT

The
sigE
gene of
Streptomyces coelicolor
A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed
sigE
deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from
Bacillus subtilis
. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The
sigE
mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that
sigE
is required for normal cell wall structure. The role of ς
E
was further investigated by analyzing the expression of
hrdD
, which is partially
sigE
dependent. The
hrdD
gene, which encodes the ς
HrdD
subunit of RNA polymerase, is transcribed from two promoters,
hrdDp
1
and
hrdDp
2
, both similar to promoters recognized by other ECF sigma factors. The activities of
hrdDp
1
and
hrdDp
2
were reduced 20- and 3-fold, respectively, in
sigE
mutants, although only
hrdDp
1
was recognized by Eς
E
in vitro. Growth on media deficient in magnesium caused the induction of both
hrdD
promoters in a
sigE
-dependent manner.
Divalent Cation Block of Inward Currents and Low-Affinity K
+
Uptake in
Saccharomyces cerevisiae
Authors: Roberts Stephen K. , Fischer Marc , Dixon Graham K. , Sanders Dale 
Published: December 2019

ABSTRACT

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a
trk1Δ trk2Δ
strain of
Saccharomyces cerevisiae
which lacks high- and moderate-affinity K
+
uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K
+
and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K
+
with Rb
+
, Cs
+
, or Na
+
only slightly modulated the magnitude of the inward current, whereas replacement with Li
+
reduced the inward current by approximately 50%, and tetraethylammonium (TEA
+
) and choline were relatively impermeant. The inward current was blocked by extracellular Ca
2+
and Mg
2+
with apparent

K
i

s (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K
+
increased the magnitude of the inward current independently of the electrochemical driving force for K
+
influx, consistent with regulation of the inward current by cytosolic K
+
. Uptake of
86
Rb
+
by intact
trk1Δ trk2Δ
cells was inhibited by extracellular Ca
2+
with a

K
i

within the range observed for the inward current. Furthermore, increasing extracellular Ca
2+
from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.
Transduction-Like Gene Transfer in the Methanogen
Methanococcus voltae
Authors: Bertani Giuseppe 
Published: December 2019

ABSTRACT

Strain PS of
Methanococcus voltae
(a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (
voltae
transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the
M. voltae
cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10
−5
(BES resistance) to a maximum of 10
−3
(histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the
Alu
I restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of
M. voltae
.
Alterations in Protein Expression Caused by the
hha
Mutation in
Escherichia coli
: Influence of Growth Medium Osmolarity
Authors: Balsalobre Carlos , Johansson Jörgen , Uhlin Bernt Eric , Juárez Antonio , Muñoa Francisco J. 
Published: December 2019

ABSTRACT

The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the
hha
mutation on the overall protein pattern of
Escherichia coli
cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the
hha
mutation. The number of proteins whose expression was altered in
hha
cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the
hha
allele, both OmpA and protein IIA
Glc
of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of the
lacZ
gene was also modified in
hha
mutants. This was the case, suggesting that at least some of the pleiotropic effects of the
hha
mutation could be caused by its effect on the catabolite repression system.