bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multidrug Efflux Pump AcrAB of
Salmonella typhimurium
Excretes Only Those β-Lactam Antibiotics Containing Lipophilic Side Chains
Authors: Nikaido Hiroshi , Basina Marina , Nguyen Vy , Rosenberg Emiko Y. 
Published: December 2019

ABSTRACT

We found that the previously reported SS-B drug-supersusceptible mutant of
Salmonella typhimurium
(S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704–712, 1984) had a mutation in the
acrAB
operon. Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to β-lactam antibiotics in the complete absence of β-lactamase. The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump. This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump. According to this mechanism, the pump successfully excretes even those β-lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer. Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically. The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type
S. typhimurium
was previously interpreted as the result of poor diffusion across the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118–132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.
Deletion of the Alternative Sigma Factor ς
B
in
Staphylococcus aureus
Reveals Its Function as a Global Regulator of Virulence Genes
Authors: Kullik Ines , Giachino Philipp , Fuchs Thomas 
Published: December 2019

ABSTRACT

A deletion of the
sigB
operon was constructed in three genetically distinct
Staphylococcus aureus
strains, and the phenotypes of the resulting mutants were analyzed. Compared to the corresponding wild-type strains, the Δ
sigB
mutants showed reduced pigmentation, accelerated sedimentation, and increased sensitivity to hydrogen peroxide during the stationary growth phase. A cytoplasmic protein missing in the Δ
sigB
mutants was identified as alkaline shock protein 23, and an extracellular protein excreted at higher levels in one of the Δ
sigB
mutants was identified as staphylococcal thermonuclease. Interestingly, most
sigB
deletion phenotypes were only seen in
S. aureus
COL and Newman and not in 8325, which was found to contain an 11-bp deletion in the regulator gene
rsbU
. Taken together, our results show that ς
B
is a global regulator which modulates the expression of several virulence factors in
S. aureus
and that laboratory strain 8325 is a ς
B
-defective mutant.
Topological Analysis of DcuA, an Anaerobic C
4
-Dicarboxylate Transporter of
Escherichia coli
Authors: Golby Paul , Kelly David J. , Guest John R. , Andrews Simon C. 
Published: December 2019

ABSTRACT

Escherichia coli
possesses three independent anaerobic C
4
-dicarboxylate transport systems encoded by the
dcuA
,
dcuB
, and
dcuC
genes. The
dcuA
and
dcuB
genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470–6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a β-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the “positive-inside rule.” Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the
lac
UV5-promoter-based plasmid, pYZ4.
Transcriptional Regulation and Evolution of Lactose Genes in the Galactose-Lactose Operon of
Lactococcus lactis
NCDO2054
Authors: Vaughan Elaine E. , Pridmore R. David , Mollet Beat 
Published: December 2019

ABSTRACT

The genetics of lactose utilization within the slow-lactose-fermenting
Lactococcus lactis
strain NCDO2054 was studied with respect to the organization, expression, and evolution of the
lac
genes. Initially the β-galactosidase gene (
lacZ
) was cloned by complementation of an
Escherichia coli
mutant on a 7-kb
Hpa
I fragment. Nucleotide sequence analysis of the complete fragment revealed part of a
gal-lac
operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order is
galK-galT-lacA-lacZ-galE
; the
gal
genes encode enzymes of the Leloir pathway for galactose metabolism, and
lacA
encodes a galactoside acetyltransferase. The
galT
and
galE
genes of
L. lactis
LM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting
L. lactis
C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening
lacA-lacZ
strain NCDO2054. Analysis of mRNA for the
gal
and
lac
genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the
lac
genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless β-glucuronidase gene (
gusA
) from
E. coli
, which identified three lactose-inducible intergenic promoters in the
gal-lac
operon. The greater similarity of the
lacA
and
lacZ
genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the
gal
genes, suggests that the genes within the
gal
operon of
L. lactis
NCDO2054 have been recently acquired. Thus, the
lacA-lacZ
genes appear to have engaged the promoters of the
gal
operon in order to direct and control their expression.
YscB of
Yersinia pestis
Functions as a Specific Chaperone for YopN
Authors: Jackson Michael W. , Day James B. , Plano Gregory V. 
Published: December 2019

ABSTRACT

Following contact with a eucaryotic cell,
Yersinia
species pathogenic for humans (
Y. pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37°C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or “contact-dependent,” secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the
yscB
gene product in the regulation of Yop secretion in
Y. pestis
. A
yscB
deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of
yscB
in
trans
completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in
yopN
eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in
Y. pestis
.
Mutation of an Alternative Sigma Factor in the Cyanobacterium
Nostoc punctiforme
Results in Increased Infection of Its Symbiotic Plant Partner,
Anthoceros punctatus
Authors: Campbell Elsie Lin , Brahamsha Bianca , Meeks John C. 
Published: December 2019

ABSTRACT

An alternative group 2 sigma factor was identified in the nitrogen-fixing, symbiotically competent cyanobacterium
Nostoc punctiforme
and designated
sigH
. Transcription of
sigH
was specifically induced within 1.5 h following exposure of
N. punctiforme
to its symbiotic plant partner,
Anthoceros punctatus
. A mutation in
sigH
resulted in a sixfold-higher initial infection of
A. punctatus
tissue without a parallel increase in nitrogen-fixing activity.
The Na
+
-Responsive
ntp
Operon Is Indispensable for Homeostatis of K
+
and Na
+
in
Enterococcus hirae
at Limited Proton Potential
Authors: Kawano Miyuki , Igarashi Kazuei , Kakinuma Yoshimi 
Published: December 2019

ABSTRACT

Enterococcus hirae
ATCC 9790 grew well in Na
+
-deficient, low-K
+
medium, but growth was inhibited by carbonylcyanide
m
-chlorophenylhydrazone (CCCP). Growth inhibition and decrease of cellular K
+
levels in the presence of CCCP were relieved by the addition of Na
+
and a high concentration of K
+
. In contrast, in the mutant defective in Na
+
-ATPase or the NtpJ component of the KtrII K
+
uptake system, CCCP-induced growth inhibition was rescued by a high concentration of K
+
but not of Na
+
. These transporters are thus indispensable for homeostatis of K
+
and Na
+
at low proton potential.
Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from
Borrelia turicatae
, a Causative Agent of Relapsing Fever
Authors: Carlyon Jason A. , Marconi Richard T. 
Published: December 2019

ABSTRACT

Borrelia turicatae
is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from
B. turicatae
and other relapsing-fever spirochetes that exhibits homology with the
rep
+
and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated
repA
, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that
repA
(or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both
Borrelia parkeri
and
B. turicatae
. Transcriptional analyses demonstrate that
repA
is expressed, albeit at low levels, during in vitro cultivation of
B. turicatae
. Transcriptional start site analysis revealed that
repA
is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of
repA
and its homologs suggest that RepA may play an important genus-wide role in the biology of the
Borrelia
.
Isolation and Characterization of High-Osmolarity-Sensitive Mutants of Fission Yeast
Authors: Aiba Hirofumi , Kawaura Ryosuke , Yamamoto Eiji , Yamada Hisami , Takegawa Kaoru , Mizuno Takeshi 
Published: December 2019

ABSTRACT

For the fission yeast
Schizosaccharomyces pombe
, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (
gpd1
+
) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive
S. pombe
mutants, including
wis1
,
sty1
, and
gpd1
. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated
hos1
,
hos2
, and
hos3
(high osmolarity sensitive) mutants. One of them, the
hos1
mutant, was characterized in detail. The
hos1
mutant was demonstrated to have a mutational lesion in the known
ryh1
+
gene, which encodes a small GTP-binding protein. Disruption of the
ryh1
+
gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the Δ
ryh1
mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of
S. pombe
.
Structure and Mechanism of Action of the Protease That Degrades Small, Acid-Soluble Spore Proteins during Germination of Spores of
Bacillus
Species
Authors: Nessi Claudio , Jedrzejas Mark J. , Setlow Peter 
Published: December 2019

ABSTRACT

The germination protease (GPR) of
Bacillus megaterium
initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P
46
) removes an ∼15-kDa C-terminal domain generating a 30-kDa species (P
30
) which is stable against further digestion. While P
30
is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P
46
to the active 41-kDa form of GPR (P
41
). Trypsin treatment of P
41
cleaves the same bond in the C-terminal part of the protein as is cleaved in the P
46
→P
30
conversion. While the ∼29-kDa species generated by trypsin treatment of P
41
is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P
41
which is 13°C lower than that for P
46
and the unfolding of P
41
at significantly lower concentrations of guanidine hydrochloride than for P
46
, are further evidence for a difference in tertiary structure between P
46
and P
41
, with P
46
presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P
46
and P
41
. The removal of ∼30% of P
46
or P
41
without significant loss in enzyme activity localized GPR’s catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P
41
by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.
Filamentous Bacteriophages of
Vibrio parahaemolyticus
as a Possible Clue to Genetic Transmission
Authors: Chang Bin , Taniguchi Hatsumi , Miyamoto Hiroshi , Yoshida Shin-ichi 
Published: December 2019

ABSTRACT

We have previously reported the isolation and characterization of two filamentous bacteriophages of
Vibrio parahaemolyticus
, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of
Vibrio cholerae
and coliphage Ff of
Escherichia coli
, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of
V. parahaemolyticus
at the distinctive region of the phage genome and were also distributed on some plasmids of
V. parahaemolyticus
and total cellular DNAs of one
Vibrio damsela
and one nonagglutinable
Vibrio
strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of
V. parahaemolyticus
strains and of genetic transmission among strains through these filamentous phages.
Expression of the Kdp ATPase Is Consistent with Regulation by Turgor Pressure
Authors: Malli Ravi , Epstein Wolfgang 
Published: December 2019

ABSTRACT

The
kdpFABC
operon of
Escherichia coli
encodes the four protein subunits of the Kdp K
+
transport system. Kdp is expressed when growth is limited by the availability of K
+
. Expression of Kdp is dependent on the products of the adjacent
kdpDE
operon, which encodes a pair of two-component regulators. Studies with
kdp-lac
fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464–468, 1981). More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H. Asha and J. Gowrishankar, J. Bacteriol. 175:4528–4537, 1993). We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured. In all cases, Kdp expression correlated with the K
+
concentration when growth began to slow. Making the reasonable but currently untestable assumptions that the reduction in growth rate by K
+
limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure.
The
Pseudomonas syringae
pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate
Authors: Charkowski Amy O. , Alfano James R. , Preston Gail , Yuan Jing , He Sheng Yang , Collmer Alan 
Published: December 2019

ABSTRACT

The host-specific plant pathogen
Pseudomonas syringae
elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the
P. syringae
genome.
hrpW
was found in a region adjacent to the
hrp
cluster in
P. syringae
pv. tomato DC3000.
hrpW
encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from
Nectria haematococca
,
Erwinia carotovora
,
Erwinia chrysanthemi
, and
Bacillus subtilis
, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in
A
230
assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the
P. syringae
pv. syringae B728a
hrpW
was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the
P. syringae
pv. tomato
hrpW
were found in other
P. syringae
pathovars,
Pseudomonas viridiflava
,
Ralstonia
(
Pseudomonas
)
solanacearum
, and
Xanthomonas campestris
. Δ
hrpZ
::
nptII
or
hrpW
::ΩSp
r
P. syringae
pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a
hrpZ hrpW
double mutant. These features of
hrpW
and its product suggest that
P. syringae
produces multiple harpins and that the target of these proteins is in the plant cell wall.
Ndd, the Bacteriophage T4 Protein That Disrupts the
Escherichia coli
Nucleoid, Has a DNA Binding Activity
Authors: Bouet Jean-Yves , Krisch Henry M. , Louarn Jean-Michel 
Published: December 2019

ABSTRACT

Early in a bacteriophage T4 infection, the phage
ndd
gene causes the rapid destruction of the structure of the
Escherichia coli
nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected
E. coli
, overexpression of the cloned
ndd
gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.
Cell Division Inhibition in
Salmonella typhimurium
Histidine-Constitutive Strains: an
ftsI
-Like Defect in the Presence of Wild-Type Penicillin-Binding Protein 3 Levels
Authors: Cano David A. , Mouslim Chakib , Ayala Juan A. , García-del Portillo Francisco , Casadesús Josep 
Published: December 2019

ABSTRACT

Histidine-constitutive (His
c
) strains of
Salmonella typhimurium
undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by His
c
strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of His
c
strains. However, the His
c
septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out.
d
-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of His
c
strains may be caused by scarcity of PBP3 substrate.
Regulation of
Escherichia coli secA
by Cellular Protein Secretion Proficiency Requires an Intact Gene X Signal Sequence and an Active Translocon
Authors: Oliver Donald , Norman Jessica , Sarker Shameema 
Published: December 2019

ABSTRACT

secA
is translationally regulated by the protein secretion proficiency state of the
Escherichia coli
cell. This regulation was explored by making signal sequence mutations in the gene upstream of
secA
, gene X, which promotes
secA
translational coupling. Gene X signal sequence mutants were constitutive for
secA
expression, while
prlA
alleles partially restored
secA
regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper
secA
regulation. Furthermore, gene X signal sequence mutations disrupted
secA
regulation only in the
cis
configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.