bluuebunny/crossref_metadata_2025 huggingface dataset:
Phage-Displayed T-Cell Epitope Grafted into Immunoglobulin Heavy-Chain Complementarity-Determining Regions: an Effective Vaccine Design Tested in Murine Cysticercosis
Authors: Manoutcharian Karen , Terrazas Luis Ignacio , Gevorkian Goar , Acero Gonzalo , Petrossian Pavel , Rodriguez Miriam , Govezensky Tzipe
Published: December 2019

A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (VH) domain with theTaenia crassicepsepitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4+and CD8+T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig VHdomains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.
The EspD Protein of Enterohemorrhagic
Escherichia coli
Is Required for the Formation of Bacterial Surface Appendages and Is Incorporated in the Cytoplasmic Membranes of Target Cells
Authors: Kresse Andreas U. , Rohde Manfred , Guzmán Carlos A.
Published: December 2019

The formation of EspA-containing surface appendages in pathogenic
Escherichia coli
strains, both enteropathogenic
E. coli
(EPEC) and Shiga toxin-producing
E. coli
strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the
esp
operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame
espD
deletion mutant of the enterohemorrhagic
E. coli
(EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933
espD
mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.
Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from
Streptomyces roseosporus
Authors: Mchenney Margaret A. , Hosted Thomas J. , Dehoff Bradley S. , Rosteck Paul R. , Baltz Richard H.
Published: December 2019

The daptomycin biosynthetic gene cluster of
Streptomyces roseosporus
was analyzed by Tn
5099
mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.
Differential Expression of the OmpF and OmpC Porin Proteins in
Escherichia coli
K-12 Depends upon the Level of Active OmpR
Authors: Lan Chung-Yu , Igo Michele M.
Published: December 2019

We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.
Identification and Cloning of
waaF
(
rfaF
) from
Bordetella pertussis
and Use To Generate Mutants of
Bordetella
spp. with Deep Rough Lipopolysaccharide
Authors: Allen Andrew G. , Isobe Tomoko , Maskell Duncan J.
Published: December 2019

A DNA locus from
Bordetella pertussis
capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in
Salmonella typhimurium
SL3789 (
rfaF511
) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by
waaF
(previously
rfaF
) in other gram-negative bacteria. Mutation of this gene in
B. pertussis
,
Bordetella parapertussis
, and
Bordetella bronchiseptica
by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in
B. parapertussis
and
B. bronchiseptica
and the first deep rough mutants of any of the bordetellae.
Stimulation of Transcription by Mutations Affecting Conserved Regions of RNA Polymerase II
Authors: Archambault Jacques , Jansma David B. , Kawasoe Jean H. , Arndt Kim T. , Greenblatt Jack , Friesen James D.
Published: December 2019

Mutations that increase the low-level transcription of the
Saccharomyces cerevisiae HIS4
gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in
SIT1
(also known as
RPO21
,
RPB1
, and
SUA8
), the gene encoding the largest subunit of RNA polymerase II (RNAPII). Here we show that
sit1
substitutions cluster in two conserved regions of the enzyme which form part of the active site. Six
sit1
mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to α-aminatin. Four
sit1
substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg
2+
ions essential for RNA catalysis. One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with
PPR2
, the gene encoding transcription elongation factor IIS. Some
sit1
substitutions affect the selection of transcriptional start sites at the
CYC1
promoter in a manner reminiscent of that of
sua8
(
sua
stands for suppression of upstream ATG) mutations. Together with previous findings which indicate that regions D and G are in close proximity to the 3′ end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the
sit1
mutations results from alteration of the RNAPII active center.
(Methyl)ammonium Transport in the Nitrogen-Fixing Bacterium
Azospirillum brasilense
Authors: Van Dommelen Anne , Keijers Veerle , Vanderleyden Jos , de Zamaroczy Miklos
Published: December 2019

An ammonium transporter of
Azospirillum brasilense
was characterized. In contrast to most previously reported putative prokaryotic NH
4
+
transporter genes,
A. brasilense amtB
is not part of an operon with
glnB
or
glnZ
which, in
A. brasilense
, encode nitrogen regulatory proteins P
II
and P
Z
, respectively. Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein. Nitrogen regulates the transcription of the
amtB
gene in
A. brasilense
by the Ntr system.
amtB
is the first gene identified in
A. brasilense
whose expression is regulated by NtrC. The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH
4
+
transport mechanism. Growth of
amtB
mutants at low ammonium concentrations is reduced compared to that of the wild type. This suggests that AmtB has a role in scavenging ammonium at low concentrations.
The Wzz (Cld) Protein in
Escherichia coli
: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity
Authors: Franco Agustin V. , Liu Dan , Reeves Peter R.
Published: December 2019

The O antigen is a polymer with a repeated unit. The chain length in most
Escherichia coli
strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values.
wzz
(
cld/rol
) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned
wzz
genes from
E. coli
strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric
wzz
genes and site-directed mutagenesis of the
wzz
gene to show that the modal value of O-antigen chain length of
E. coli
O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in
wzz
. It is concluded that the O-antigen chain length heterogeneity in
E. coli
strains is the result of amino acid sequence variation of the Wzz protein.
In Vitro and In Vivo Oxidation of Methionine Residues in Small, Acid-Soluble Spore Proteins from
Bacillus
Species
Authors: Hayes Christopher S. , Illades-Aguiar Berenice , Casillas-Martinez Lilliam , Setlow Peter
Published: December 2019

Methionine residues in α/β-type small, acid-soluble spore proteins (SASP) of
Bacillus
species were readily oxidized to methionine sulfoxide in vitro by
t
-butyl hydroperoxide (tBHP) or hydrogen peroxide (H
2
O
2
). These oxidized α/β-type SASP no longer bound to DNA effectively, but DNA binding protected α/β-type SASP against methionine oxidation by peroxides in vitro. Incubation of an oxidized α/β-type SASP with peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues back to methionine, restored the α/β-type SASP’s ability to bind to DNA. Both tBHP and H
2
O
2
caused some oxidation of the two methionine residues of an α/β-type SASP (SspC) in spores of
Bacillus subtilis
, although one methionine which is highly conserved in α/β-type SASP was only oxidized to a small degree. However, much more methionine sulfoxide was generated by peroxide treatment of spores carrying a mutant form of SspC which has a lower affinity for DNA. MsrA activity was present in wild-type
B. subtilis
spores. However,
msrA
mutant spores were no more sensitive to H
2
O
2
than were wild-type spores. The major mechanism operating for dealing with oxidative damage to α/β-type SASP in spores is DNA binding, which protects the protein’s methionine residues from oxidation both in vitro and in vivo. This may be important in vivo since α/β-type SASP containing oxidized methionine residues no longer bind DNA well and α/β-type SASP-DNA binding is essential for long-term spore survival.
Generation ofEscherichia coliO9a Serotype, a Subtype ofE. coliO9, by Transfer of thewb* Gene Cluster ofKlebsiellaO3 intoE. colivia Recombination
Authors: Sugiyama Tsuyoshi , Kido Nobuo , Kato Yutaka , Koide Naoki , Yoshida Tomoaki , Yokochi Takashi
Published: December 2019

Genetic characterization of thewb* gene in a series ofEscherichia coliandKlebsiellastrains possessing the mannose homopolymer as the O-specific polysaccharide was carried out. The partial nucleotide sequences and PCR-restriction fragment length polymorphism analysis suggested thatE. coliserotype O9a, a subtype ofE. coliO9, might have been generated by the insertion of theKlebsiellaO3wb* gene into a certainE. colistrain.
Menaquinone (Vitamin K
2
) Biosynthesis: Localization and Characterization of the
menA
Gene from
Escherichia coli
Authors: Suvarna K. , Stevenson D. , Meganathan R. , Hudspeth M. E. S.
Published: December 2019

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The
menA
gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the
Escherichia coli
genome between
cytR
and
glpK
. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a
menA
mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the
orf
-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the
menA
product into the
orf
-complemented
menA
mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.
Mutational Analysis of the Phosphate-Binding Loop of
Rhizobium meliloti
DctD, a ς
54
-Dependent Activator
Authors: Gao Yan , Wang Ying-Kai , Hoover Timothy R.
Published: December 2019

The phosphate-binding loop of ς
54
-dependent activators is thought to participate in ATP binding and/or hydrolysis. Alanine substitutions at positions 3, 4, 6, 7, and 8 of this motif in
Rhizobium meliloti
DctD disrupted transcriptional activation and ATP hydrolysis. Interestingly, substitution of alanine at position 7 also affected DNA binding.
Activation and Repression of Transcription at the Double Tandem Divergent Promoters for the
xylR
and
xylS
Genes of the TOL Plasmid of
Pseudomonas putida
Authors: Marqués Silvia , Gallegos María-Trinidad , Manzanera Maximino , Holtel Andreas , Timmis Kenneth N. , Ramos Juan L.
Published: December 2019

The
xylR
and
xylS
genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive ς
70
-dependent tandem promoters that drive expression of
xylR
, while expression of the
xylS
gene is driven from Ps2, a constitutive ς
70
-dependent promoter, and by the regulatable ς
54
class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions −2 to −30 (−2/−30 region) and the −137/−156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a ς
54
-deficient
Pseudomonas putida
background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing ς
54
modulates expression from the downstream Ps2 ς
70
-dependent promoter. In a
P. putida
IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the ς
54
-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing ς
54
at the upstream promoter may modulate expression from the Ps2 promoter.
The Developmentally Regulatedalb1Gene ofAspergillus fumigatus: Its Role in Modulation of Conidial Morphology and Virulence
Authors: Tsai Huei-Fung , Chang Yun C. , Washburn Ronald G. , Wheeler Michael H. , Kwon-Chung K. J.
Published: December 2019

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene,alb1, which is required for conidial pigmentation. Thealb1gene encodes a putative polyketide synthase, and disruption ofalb1resulted in an albino conidial phenotype. Expression ofalb1is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. Thealb1mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating thatalb1is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that thealb1disruptant exhibited a smooth conidial surface, whereas complementation of thealb1deletion restored the echinulate wild-type surface. Disruption ofalb1resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of thealb1disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. Thealb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, thealb1disruptant had a statistically significant loss of virulence compared to the wild-type andalb1-complemented strains in a murine model. These results suggest that disruption ofalb1causes pleiotropic effects on conidial morphology and fungal virulence.
Target Choice and Orientation Preference of the Insertion Sequence IS
903
Authors: Hu Wen-Yuan , Derbyshire Keith M.
Published: December 2019

We have examined the targeting preference of the bacterial insertion element IS
903
by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38. Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer. Within these regions, many different sites were used for insertion; however, there were a few sites that IS
903
inserted into more than once. Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target. This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target. We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment. Also, we observed a very strong preference for insertion in a single orientation in pUB307. We examined the possibility that either DNA replication from the origin of vegetative replication,
oriV
, or the origin of transfer,
oriT
, might determine this orientation effect. We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect. This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon.
Identification and Characterization of Two Quiescent Porin Genes,
nmpC
and
ompN
, in
Escherichia coli
B
E
Authors: Prilipov Alexej , Phale Prashant S. , Koebnik Ralf , Widmer Christine , Rosenbusch Jurg P.
Published: December 2019

The genomic DNA of the B
E
strain of
Escherichia coli
has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the
ompC
,
ompF
, and
phoE
porin genes. The two genes were cloned and sequenced. One of them, designated
ompN
, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from
E. coli
K-12. The second DNA fragment detected corresponds to the
nmpC
gene, which, due to an insertion of an IS
1
element in its coding region, is not expressed in
E. coli
B
E
.
Genetic Recombination in
Bacillus subtilis
168: Effects of
recU
and
recS
Mutations on DNA Repair and Homologous Recombination
Authors: Fernández Silvia , Sorokin Alexei , Alonso Juan C.
Published: December 2019

Bacillus subtilis
recombination-deficient mutants were constructed by inserting a selectable marker (
cat
gene) into the
yppB
and
ypbC
coding regions. The
yppB
:
cat
and
ypbC
:
cat
null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec
+
B. subtilis
strain. The
yppB
gene complemented the defect of the
recG40
strain.
yppB
and
ypbC
and their respective null alleles were termed “
recU
” and “
recU1
” (
recU
:
cat
) and “
recS
” and “
recS1
” (
recS
:
cat
), respectively. The
recU
and
recS
mutations were introduced into
rec
-deficient strains representative of the α (
recF
), β (
addA5 addB72
), γ (
recH342
), and ɛ (
recG40
) epistatic groups. The
recU
mutation did not modify the sensitivity of
recH
cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in
recH
cells. The
recS
mutation did not modify the sensitivity of
addAB
cells to DNA-damaging agents, and it marginally affected
recF
,
recH
, and
recU
cells. The
recS
mutation markedly reduced (about 250-fold) intermolecular recombination in
recH
cells, and there were reductions of 10- to 20-fold in
recF
,
addAB
, and
recU
cells. Intramolecular recombination was blocked in
recS recF
,
recS addAB
, and
recS recU
cells. RecU and RecS have no functional counterparts in
Escherichia coli
. Altogether, these data indicate that the
recU
and
recS
proteins are required for DNA repair and intramolecular recombination and that the
recF
(α epistatic group),
addAB
(β),
recH
(γ),
recU
(ɛ), and
recS
genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed
recS
within a new group, termed “ζ.”
tetA
(L) Mutants of a Tetracycline-Sensitive Strain of
Bacillus subtilis
with the Polynucleotide Phosphorylase Gene Deleted
Authors: Bechhofer David H. , Stasinopoulos Stan J.
Published: December 2019

A
Bacillus subtilis
strain with the polynucleotide phosphorylase gene deleted was sensitive for growth in the presence of tetracycline. This strain was used to select for tetracycline-resistant mutants. A point mutation in the
tetA
(L) promoter and a spontaneously occurring
tetA
(L) gene copy number mutant were characterized.
A Periplasmic and Extracellular
c
-Type Cytochrome of
Geobacter sulfurreducens
Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria
Authors: Seeliger Sabine , Cord-Ruwisch Ralf , Schink Bernhard
Published: December 2019

An extracellular electron carrier excreted into the growth medium by cells of
Geobacter sulfurreducens
was identified as a
c
-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes
c
of
Desulfovibrio salexigens
and
Desulfuromonas acetoxidans
. The
G. sulfurreducens
cytochrome
c
reduced ferrihydrite (Fe(OH)
3
), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly.
G. sulfurreducens
reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate.
Wolinella succinogenes
was able to reduce externally provided cytochrome
c
of
G. sulfurreducens
with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which
G. sulfurreducens
reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by
W. succinogenes
in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.
Temperature Regulation of Heat-Labile Enterotoxin (LT) Synthesis in
Escherichia coli
Is Mediated by an Interaction of H-NS Protein with the LT A-Subunit DNA
Authors: Trachman Julie D. , Maas Werner K.
Published: December 2019

Protein and mRNA levels of heat-labile enterotoxin (LT) of
Escherichia coli
are highest at 37°C, and they decrease gradually as temperature is decreased. This temperature effect is eliminated in an Hns
−
mutant. Deletion of portions of DNA coding for the LT A subunit also results in an increase in LT expression at low temperatures, suggesting that the H-NS protein causes inhibition of transcription at low temperatures by interacting with the LT A-subunit DNA. The region that interacts with H-NS is referred to as the downstream regulatory element (DRE). Plasmids in an
hns
strain from which the DRE has been deleted still produce elevated levels of LT at 18°C, suggesting that intact DRE is not required for transcription from the LT promoter.
Opposing Roles of the
Staphylococcus aureus
Virulence Regulators, Agr and Sar, in Triton X-100- and Penicillin-Induced Autolysis
Authors: Fujimoto David F. , Bayles Kenneth W.
Published: December 2019

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the
Staphylococcus aureus
virulence factor regulatory genes,
agr
and
sar
, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest that
agr
and
sar
exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.
Biosynthesis of Di-
myo
-Inositol-1,1′-Phosphate, a Novel Osmolyte in Hyperthermophilic Archaea
Authors: Chen Liangjing , Spiliotis Elias T. , Roberts Mary F.
Published: December 2019

Biosynthesis of di-
myo
-inositol-1,1′-phosphate (DIP) is proposed to occur with
myo
-inositol and
myo
-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with
myo
-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of
d
-glucose-6-phosphate to
myo
-inositol. This conversion requires two key enzymes:
l
-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using
31
P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of
l
-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with
myo
-inositol to generate DIP has also been detected in
Methanococcus igneus
.
13
C labeling studies with [2,3-
13
C]pyruvate and [3-
13
C]pyruvate were used to examine this pathway in
M. igneus
. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.
Dentilisin Activity Affects the Organization of the Outer Sheath ofTreponema denticola
Authors: Ishihara Kazuyuki , Kuramitsu Howard K. , Miura Tadashi , Okuda Katsuji
Published: December 2019

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced byTreponema denticola. The gene,prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence ofT. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with aprtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant withFusobacterium nucleatumwas enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath ofT. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity ofT. denticola.
Chemical Structure of Lipid A Isolated fromFlavobacterium meningosepticumLipopolysaccharide
Authors: Kato Hitomi , Haishima Yuji , Iida Takatoshi , Tanaka Akira , Tanamoto Ken-ichi
Published: December 2019

The chemical structure of the lipid A of the lipopolysaccharide component isolated fromFlavobacterium meningosepticumIFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the free lipid A: a β (1→6)-linked 2-amino-2-deoxy-d-glucose, which is usually present in enterobacterial lipid A’s, and a 2-amino-6-O-(2,3-diamino-2,3-dideoxy-β-d-glucopyranosyl)-2-deoxy-d-glucose, in a molar ratio of 1.00:0.35. Both backbones were α-glycosidically phosphorylated in position 1, and the hydroxyl groups at positions 4, 4′, and 6′ were unsubstituted. Liquid secondary ion-mass spectrometry revealed a pseudomolecular ion atm/z1673 [M-H]−as a major monophosphoryl lipid A component carrying five acyl groups. Fatty acid analysis showed that the lipid A contained 1 mol each of amide-linked (R)-3-OH iC17:0, ester-linked (R)-3-OH iC15:0, amide-linked (R)-3-O-(iC15:0)-iC17:0, and both amide- and ester-linked (R)-3-OH C16:0. Fatty acid distribution analyses using several mass spectrometry determinations demonstrated that the former two constituents were distributed on positions 2 and 3 of the reducing terminal unit of the backbones and that the latter two were attached to the 2′ and 3′ positions in the nonreducing terminal residue.
Bacterial SOS Checkpoint Protein SulA Inhibits Polymerization of Purified FtsZ Cell Division Protein
Authors: Trusca Dorina , Scott Solomon , Thompson Chris , Bramhill David
Published: December 2019

Cell division of
Escherichia coli
is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.
Both the
fipA
Gene of pKM101 and the
pifC
Gene of F Inhibit Conjugal Transfer of RP1 by an Effect on
traG
Authors: Santini Joanne M. , Stanisich Vilma A.
Published: December 2019

The mechanisms by which gene products inhibit the conjugal transfer of IncP plasmids (e.g., RP1) have been little studied. We have isolated and characterized one such gene,
fipA
(624 nucleotides), from the
Sma
I (14.8 kb)-
Aat
II (15.6 kb) region of pKM101(IncN). This gene, which is also conserved in other IncN plasmids, is transcribed in an anticlockwise direction, probably as part of a transfer operon that includes
traHI
. The FipA protein (24 kDa) appears to be cytoplasmic and, when expressed from a multicopy plasmid, retards the growth of
Escherichia coli
WP2. The mode of action of
fipA
was compared with that of the apparently unrelated
pifC
gene from F(IncFI). Both genes inhibit the transfer of IncPα and IncPβ plasmids but to different degrees. They also inhibit the mobilization of RSF1010 (which requires the RP1 pilus genes and
traG
) but not of CloDF13 (which encodes a
traG
homolog). Evidence that
traG
was the specific target of inhibition was obtained in an artificial system in which cloned
traG
was used to enhance RSF1010 mobilization via the N pilus system. Such enhancement did not occur in the presence of
fipA
or
pifC
. The availability of an in vivo assay of PifC enabled us to show that F
pif
operon expression increased in cells carrying F′
lac
and
traG
, but only if the
traG
coding sequence was intact. This finding suggested that conjugal inhibition of RP1 was most likely due to a PifC-TraG protein interaction. On phenotypic grounds inhibition of
traG
by
fipA
is also likely to occur posttranscriptionally. Whether or not the selection of
traG
as the inhibition target is an evolutionary tactic to limit the spread of P plasmids, we anticipate that
fipA
and
pifC
will prove useful in further investigation of the conjugal roles of
traG
and its homologs.
A Molecular Switch Controlling Competence and Motility: Competence Regulatory Factors ComS, MecA, and ComK Control ς
D
-Dependent Gene Expression in
Bacillus subtilis
Authors: Liu Jiajian , Zuber Peter
Published: December 2019

Bacillus subtilis
, like many bacteria, will choose among several response pathways when encountering a stressful environment. Among the processes activated under growth-restricting conditions are sporulation, establishment of motility, and competence development. Recent reports implicate ComK and MecA-ClpC as part of a system that regulates both motility and competence development. MecA, while negatively controlling competence by inhibiting ComK, stimulates ς
D
-dependent transcription of genes that function in motility and autolysin production. Both ComK-dependent and -independent pathways have been proposed for MecA’s role in the regulation of motility. Mutations in
mecA
reduce the transcription of
hag
. encoding flagellin, and are partially suppressed by
comK
in both medium promoting motility and medium promoting competence. Reduced ς
D
levels are observed in
mecA
mutants grown in competence medium, but no change in ς
D
concentration is detected in a
comK
mutant. The
comF
operon, transcription of which requires ComK, is located immediately upstream of the operon that contains the
flgM
gene, encoding the ς
D
-specific antisigma factor. An insertion mutation that disrupts the putative
comF-flgM
transcription unit confers a phenotype identical to that of the
comK
mutant with respect to
hag-lacZ
expression. Expression of a
flgM-lacZ
operon fusion is reduced in both
sigD
and
comK
mutant cells but is abolished in the
sigD comK
double mutant. Reverse transcription-PCR examination of the
comF-flgM
transcript indicates that readthrough from
comF
into the
flgM
operon is dependent on ComK. ComK negatively controls the transcription of
hag
by stimulating the transcription of
comF-flgM
, thereby increasing the production of the FlgM antisigma factor that inhibits ς
D
activity. There likely exists another
comK
-independent mechanism of
hag
transcription that requires
mecA
and possibly affects the ς
D
concentration in cells undergoing competence development.
Involvement of the
gapA
- and
epd
(
gapB
)-Encoded Dehydrogenases in Pyridoxal 5′-Phosphate Coenzyme Biosynthesis in
Escherichia coli
K-12
Authors: Yang Yong , Zhao Genshi , Man Tsz-Kwong , Winkler Malcolm E.
Published: December 2019

We show that
epd
(
gapB
) mutants lacking an erythrose 4-phosphate (E4P) dehydrogenase are impaired for growth on some media and contain less pyridoxal 5′-phosphate (PLP) and pyridoxamine 5′-phosphate (PMP) than their
epd
+
parent. In contrast to a previous report, we found that
gapA epd
double mutants lacking the glyceraldehyde 3-phosphate and E4P dehydrogenases are auxotrophic for pyridoxine. These results implicate the GapA and Epd dehydrogenases in de novo PLP and PMP coenzyme biosynthesis.
Importance of Glutathione for Growth and Survival of
Escherichia coli
Cells: Detoxification of Methylglyoxal and Maintenance of Intracellular K
+
Authors: Ferguson G. P. , Booth I. R.
Published: December 2019

The role of the tripeptide glutathione in the growth and survival of
Escherichia coli
cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to
E. coli
cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K
+
pool and survival of exposure to methylglyoxal.
The
carB
Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from
Lactococcus lactis
Is Transcribed Monocistronically
Authors: Martinussen Jan , Hammer Karin
Published: December 2019

The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (
carB
) in
Lactococcus lactis
is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis,
L. lactis
is shown to possess only one
carB
gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the
carB
gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the
carB
gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.
Transcriptional Regulation of
CLN3
Expression by Glucose in
Saccharomyces cerevisiae
Authors: Parviz Fereshteh , Hall Duane D. , Markwardt David D. , Heideman Warren
Published: December 2019

In
Saccharomyces cerevisiae
, the transition from the G
1
phase of the mitotic cycle into S phase is controlled by a set of G
1
cyclins that regulate the activity of the protein kinase encoded by
CDC28
. Yeast cells regulate progress through the G
1
/S boundary in response to nutrients, moving quickly through G
1
in glucose medium and more slowly in poorer medium. We have examined connections between glucose and the level of the message encoding Cln3, a G
1
cyclin. We found that glucose positively regulates
CLN3
mRNA levels through a set of repeated AAGAAAAA (A
2
GA
5
) elements within the
CLN3
promoter. Mutations in these sequences reduce both transcriptional activation and specific interaction between
CLN3
promoter elements and proteins in yeast extracts. Creation of five point mutations, replacing the G’s within these repeats with T’s, in the
CLN3
promoter substantially reduces
CLN3
expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose.
Peptidoglycan Structural Dynamics during Germination of
Bacillus subtilis
168 Endospores
Authors: Atrih Abdelmadjid , Zöllner Peter , Allmaier Günter , Williamson Michael P. , Foster Simon J.
Published: December 2019

Peptidoglycan structural dynamics during endospore germination of
Bacillus subtilis
168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant
l
-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of
B. subtilis
168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic δ-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.
Cloning, Expression, and Catabolite Repression of a Gene Encoding β-Galactosidase of
Bacillus megaterium
ATCC 14581
Authors: Shaw Gwo-Chyuan , Kao Hsun-Sheng , Chiou Chih-Yung
Published: December 2019

A gene encoding β-galactosidase, designated
mbgA
, was isolated from
Bacillus megaterium
ATCC 14581. Chromosomal β-galactosidase production could be dramatically induced by lactose but not by isopropyl-β-
d
-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of
mbgA
in the
B. megaterium
chromosome resulted in loss of lactose-inducible β-galactosidase production. A 27-bp inverted repeat was found to overlap the
mbgA
promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).
The
panE
Gene, Encoding Ketopantoate Reductase, Maps at 10 Minutes and Is Allelic to
apbA
in
Salmonella typhimurium
Authors: Frodyma Michael E. , Downs Diana
Published: December 2019

In
Salmonella typhimurium
, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway. The
apbA
gene was the first locus defined as required for function of the APB pathway (D. M. Downs and L. Petersen, J. Bacteriol. 176:4858–4864, 1994). Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid. This activity had previously been associated with the pantothenate biosynthetic gene
panE
. Although previous reports placed
panE
at 87 min on the
Escherichia coli
chromosome, we show herein that
apbA
and
panE
are allelic and map to 10 min on both the
S. typhimurium
and
E. coli
chromosomes. Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.
Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Ghosh Mousumi , Grunden Amy M. , Dunn Dianne M. , Weiss Robert , Adams Michael W. W.
Published: December 2019

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon
Pyrococcus furiosus
by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co
2+
ions (

K
d

, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co
2+
could be replaced by Mn
2+
(resulting in a 25% decrease in activity) but not by Mg
2+
, Ca
2+
, Fe
2+
, Zn
2+
, Cu
2+
, or Ni
2+
. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the
P. furiosus
genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in
Escherichia coli
and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from
P. furiosus
. Furthermore, the

K
m

values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater
V
max
value than the native protein. The amino acid sequence of
P. furiosus
prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The
P. furiosus
enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.