bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific In Vivo Labeling of Cell Surface-Exposed Protein Loops: Reactive Cysteines in the Predicted Gating Loop Mark a Ferrichrome Binding Site and a Ligand-Induced Conformational Change of theEscherichia coliFhuA Protein
Authors: Bös Christoph , Lorenzen Dirk , Braun Volkmar
Published: December 2019

The FhuA protein ofEscherichia coliK-12 transports ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5, φ80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It is thought that the proteins TonB, ExbB, and ExbD function as a coupling device between the cytoplasmic membrane and the outer membrane. Excision of 34 residues from FhuA, tentatively designated the gating loop, converts FhuA into a permanently open channel. FhuA contains two disulfide bridges, one in the gating loop and one close to the C-terminal end. Reduction of the disulfide bridges results in a low in vivo reaction of the cysteines in the gating loop and no reaction of the C-terminal cysteines with biotin-maleimide, as determined by streptavidin-β-galactosidase bound to biotin. In this study we show that a cysteine residue introduced into the gating loop by replacement of Asp-336 displayed a rather high reactivity and was used to monitor structural changes in FhuA upon binding of ferrichrome. Flow cytometric analysis revealed fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by ferrichrome, but ferrichrome quenching was negligible. It is concluded that binding of ferrichrome causes a conformational change of the gating loop and that Cys-347 is part of or close to the ferrichrome binding site. Fluorescence quenching was independent of the TonB activity. The newly introduced cysteines and the replacement of the existing cysteines by serine did not alter sensitivity of cells to the FhuA ligands tested (T5, φ80, T1, colicin M, and albomycin) and fully supported growth on ferrichrome as the sole iron source. Since cells ofE. coliK-12 display no reactivity to thiol reagents, newly introduced cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.
Modulation of
Escherichia coli
Adenylyl Cyclase Activity by Catalytic-Site Mutants of Protein IIA
Glc
of the Phosphoenolpyruvate:Sugar Phosphotransferase System
Authors: Reddy Prasad , Kamireddi Madhavi
Published: December 2019

It is demonstrated here that in
Escherichia coli
, the phosphorylated form of the glucose-specific phosphocarrier protein IIA
Glc
of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA
Glc
has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIA
Glc
phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA
Glc
were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIA
Glc
and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr∼P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIA
Glc
was phosphorylated by HPr∼P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr∼P, yet the H75E mutant of IIA
Glc
was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr∼P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIA
Glc
structure and the disposition of the negative charge in the modeled glutamate mutants.
Analysis of
Bacillus subtilis tagAB
and
tagDEF
Expression during Phosphate Starvation Identifies a Repressor Role for PhoP∼P
Authors: Liu Wei , Eder Stephen , Hulett F. Marion
Published: December 2019

The
tagAB
and
tagDEF
operons, which are adjacent and divergently transcribed, encode genes responsible for cell wall teichoic acid synthesis in
Bacillus subtilis
. The
Bacillus
data presented here suggest that PhoP and PhoR are required for direct repression of transcription of the two operons under phosphate starvation conditions but have no regulatory role under phosphate-replete conditions. These data identify for the first time that PhoP∼P has a negative role in Pho regulon gene regulation.
Chimeric Chemoreceptors in
Escherichia coli
: Signaling Properties of Tar-Tap and Tap-Tar Hybrids
Authors: Weerasuriya Siromi , Schneider Brian M. , Manson Michael D.
Published: December 2019

The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of
Escherichia coli
together mediate chemotactic responses to dipeptides. Tap is a low-abundance receptor. It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr. Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run). To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains. The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator. Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain. Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap. The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability. The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation. However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody. Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg. The hybrid, truncated, and extended receptors exhibit various levels of methylation. However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected. We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.
Attachment of
Vibrio alginolyticus
to Glass Surfaces Is Dependent on Swimming Speed
Authors: Kogure Kazuhiro , Ikemoto Eiko , Morisaki Hisao
Published: December 2019

The attachment of
Vibrio alginolyticus
to glass surfaces was investigated with special reference to the swimming speed due to the polar flagellum. This bacterium has two types of flagella, i.e., one polar flagellum and numerous lateral flagella. The mutant YM4, which possesses only the polar flagellum, showed much faster attachment than the mutant YM18, which does not possess flagella, indicating that the polar flagellum plays an important role. The attachment of YM4 was dependent on Na
+
concentration and was specifically inhibited by amiloride, an inhibitor of polar flagellum rotation. These results are quite similar to those for swimming speed obtained under the same conditions. Observations with other mutants showed that chemotaxis is not critical and that the flagellum does not act as an appendage for attachment. From these results, it is concluded that the attachment of
V. alginolyticus
to glass surfaces is dependent on swimming speed.
Involvement of Outer Membrane Protein TolC, a Possible Member of the
mar-sox
Regulon, in Maintenance and Improvement of Organic Solvent Tolerance of
Escherichia coli
K-12
Authors: Aono Rikizo , Tsukagoshi Norihiko , Yamamoto Mami
Published: December 2019

Escherichia coli
mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible
mar-rob-sox
box sequence was found upstream of the
tolC
gene. These findings suggest that
tolC
is a member of the
mar-sox
regulon responsive to stress conditions. When a defective
tolC
gene was transferred to
n
-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type
tolC
gene. Therefore, it is evident that TolC is essential for
E. coli
to maintain organic solvent tolerance.
The StpA Protein Functions as a Molecular Adapter To Mediate Repression of the
bgl
Operon by Truncated H-NS in
Escherichia coli
Authors: Free Andrew , Williams Roy M. , Dorman Charles J.
Published: December 2019

The mechanism of repression of the β-glucoside utilization (
bgl
) operon of
Escherichia coli
by a carboxy-terminally truncated derivative of the nucleoid-associated protein H-NS which is defective in DNA binding was investigated. The DNA-binding function of the H-NS-like protein StpA was found to be necessary for repression, which is consistent with a role for StpA as a DNA-binding adapter for mutant derivatives of H-NS.
An ABC Transporter System of
Yersinia pestis
Allows Utilization of Chelated Iron by
Escherichia coli
SAB11
Authors: Bearden Scott W. , Staggs Teanna M. , Perry Robert D.
Published: December 2019

The acquisition of iron is an essential component in the pathogenesis of
Yersinia pestis
, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of
Y. pestis
KIM6+ was used for transduction of an
Escherichia coli
mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb
Bam
HI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(
o
-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated
yfe
, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the
yfe
locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon,
yfeABCD
, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed
yfeE
gene may be located in the cell envelope, since a
yfeE
::Tn
phoA
fusion is PhoA
+
. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.
Isolation of
Rhodospirillum centenum
Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis
Authors: Jiang Ze-Yu , Rushing Brenda G. , Bai Yong , Gest Howard , Bauer Carl E.
Published: December 2019

The purple photosynthetic bacterium
Rhodospirillum centenum
is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn
5
-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective
R. centenum
swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.
The
spoIIE
Locus Is Involved in the Spo0A-Dependent Switch in the Location of FtsZ Rings in
Bacillus subtilis
Authors: Khvorova Anastasia , Zhang Ling , Higgins Michael L. , Piggot Patrick J.
Published: December 2019

A switch in the location of FtsZ ring structures from medial to polar is one of the earliest morphological indicators of sporulation in
Bacillus subtilis
. This switch can be artificially caused during vegetative growth by induction of an active form, Sad67, of the transcription regulator, Spo0A (P. A. Levin and R. Losick, Genes Dev. 10:478–488, 1996). We have used immunofluorescence microscopy to show that the switch in FtsZ ring location during vegetative growth caused by Sad67 induction is blocked by a
spoIIE
deletion mutation. The
spoIIE
mutation also impaired polar FtsZ ring formation during sporulation. These results suggest that SpoIIE mediates the Spo0A-directed formation of polar FtsZ rings.
Insertional Inactivation of Genes Encoding the Crystalline Inclusion Proteins of
Photorhabdus luminescens
Results in Mutants with Pleiotropic Phenotypes
Authors: Bintrim Scott B. , Ensign Jerald C.
Published: December 2019

The entomopathogenic bacterium
Photorhabdus luminescens
exhibits phase variation when cultured in vitro. The variant forms of
P. luminescens
are pleiotropic and are designated phase I and phase II variants. One of the characteristic phenotypes of phase I cells is the production of two types of intracellular protein inclusions. The genes encoding the protein monomers that form these inclusions, designated
cipA
and
cipB
, were cloned and characterized.
cipA
and
cipB
encode hydrophobic proteins of 11,648 and 11,308 Da, respectively. The deduced amino acid sequences of CipA and CipB have no significant amino acid sequence similarity to any other known protein but have 25% identity and 49% similarity to each other. Insertional inactivation of
cipA
or
cipB
in phase I cells of
P. luminescens
produced mutants that differ from phase I cells in bioluminescence, the pattern and activities of extracellular products, biochemical traits, adsorption of dyes, and ability to support nematode growth and reproduction. In general, the
cip
mutants were phenotypically more similar to each other than to either phase I or phase II variants.
Sequence Diversity, Predicted Two-Dimensional Protein Structure, and Epitope Mapping of Neisserial Opa Proteins
Authors: Malorny Burkhard , Morelli Giovanna , Kusecek Barica , Kolberg Jan , Achtman Mark
Published: December 2019

The sequence diversity of 45 Opa outer membrane proteins from
Neisseria meningitidis
,
Neisseria gonorrhoeae
,
Neisseria sicca
, and
Neisseria flava
indicates that horizontal genetic exchange of
opa
alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane β-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.
Purification of an Extracellular Signaling Molecule Involved in Production of Aerial Mycelium by
Streptomyces coelicolor
Authors: Nodwell Justin R. , Losick Richard
Published: December 2019

We have extensively purified a factor from conditioned medium that restores aerial mycelium formation to a mutant of
Streptomyces coelicolor
that is defective in morphological differentiation. Response to this factor is shown to depend on the presence of the BldK oligopeptide import system. We suggest that this substance acts at the first step in a putative cascade of developmental regulatory signals.
Characterization of Mutants Devoid of Neutral Trehalase Activity in the Fission Yeast
Schizosaccharomyces pombe
: Partial Protection from Heat Shock and High-Salt Stress
Authors: Cansado Jose , Soto Teresa , Fernandez Juana , Vicente-Soler Jero , Gacto Mariano
Published: December 2019

Exposure of cells of
Schizosaccharomyces pombe
to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose. We constructed
S. pombe
mutants lacking neutral trehalase activity by gene replacement at the newly defined
ntp1
+
locus. Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media. Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation of
ntp1
+
in response to both stresses.
The LysR-Type Transcriptional Regulator CbbR Controlling Autotrophic CO
2
Fixation by
Xanthobacter flavus
Is an NADPH Sensor
Authors: van Keulen G. , Girbal L. , van den Bergh E. R. E. , Dijkhuizen L. , Meijer W. G.
Published: December 2019

Autotrophic growth of
Xanthobacter flavus
is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the
cbb
and
gap-pgk
operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the
cbb
and
gap-pgk
operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in
Escherichia coli
and subsequently purified to homogeneity. CbbR of
X. flavus
is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 μM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparent
K

d
NADPH

of CbbR was determined to be 75 μM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64° bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9°. From the results of these in vitro experiments, we conclude that CbbR responds to NADPH. The in vivo regulation of the
cbb
and
gap-pgk
operons may therefore be regulated by the intracellular concentration of NADPH.
Altered
srf
Expression in
Bacillus subtilis
Resulting from Changes in Culture pH Is Dependent on the Spo0K Oligopeptide Permease and the ComQX System of Extracellular Control
Authors: Cosby W. Mark , Vollenbroich Dirk , Lee Oh Hyoung , Zuber Peter
Published: December 2019

The expression of the
srf
operon of
Bacillus subtilis
, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in
srf
expression and surfactin production. This apparent pH-dependent induction of
srf
expression required
spo0K
, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates
srf
expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent
srf
transcription, were required for optimal expression of
srf
at low and high pHs, but ComP was not required for pH-dependent
srf
induction. The known negative regulators of
srf
, RapC and CodY, were found not to function significantly in pH-dependent
srf
expression. Late-growth culture supernatants at low pH were not active in inducing
srf
expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the
srf
-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of
srf-lacZ
at neutral pH. The results suggest that
srf
expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.
IroN, a Novel Outer Membrane Siderophore Receptor Characteristic of
Salmonella enterica
Authors: Bäumler Andreas J. , Norris Tracy L. , Lasco Todd , Voigt Wolfgang , Reissbrodt Rolf , Rabsch Wolfgang , Heffron Fred
Published: December 2019

Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize
iroN
, a gene located downstream of the
iroBC
operon in the
iroA
locus of
Salmonella enterica
serotype Typhi. Like
iroBC
, the
iroN
gene is present in all phylogenetic lineages of
S. enterica
but is absent from closely related species such as
Salmonella bongori
or
Escherichia coli
. Comparison of the deduced amino acid sequence of
iroN
with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in
S. enterica
and expression in
E. coli
identified a 78-kDa outer membrane protein as the
iroN
gene product. When introduced into an
E. coli fepA cir fiu aroB
mutant on a cosmid,
iroN
mediated utilization of structurally related catecholate siderophores, including
N
-(2,3-dihydroxybenzoyl)-
l
-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-
N
,6-
N
-bis(2,3-dihydroxybenzoyl)-
l
-lysine, 2-
N
,6-
N
-bis(2,3-dihydroxybenzoyl)-
l
-lysine amide, and enterochelin. These results suggest that the
iroA
locus functions in iron acquisition in
S. enterica.
PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in
Acinetobacter
Authors: Gerischer Ulrike , Segura Ana , Ornston L. Nicholas
Published: December 2019

The
Acinetobacter pcaIJFBDKCHG
operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are
qui
and
pob
genes encoding sets of enzymes that convert quinate and
p
-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the
pca-qui-pob
cluster was
pobR
, which encodes a transcriptional activator that responds to
p
-hydroxybenzoate and activates transcription of
pobA
. The
pca
and
qui
genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from
pcaI
and prevented expression of the
pca
genes. Sequencing of wild-type DNA extending from the translational start of
pcaI
through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated
pcaU
. Downstream from
pcaU
are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of
pcaU
reduced the induced expression of
pca
structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in
Escherichia coli
and shown to bind to a portion of the
pcaI-pcaU
intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the
pob
operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The
pob
and
pca
systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking
pca
gene expression was located in the promoter for the
pca
operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for
Pseudomonas putida
PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream from
Acinetobacter pcaU
contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in the
pcaU-pcaI
intergenic region.
Characterization of the Gene Cassette Required for Biosynthesis of the (α1→6)-Linked
N
-Acetyl-
d
-Mannosamine-1-Phosphate Capsule of Serogroup A
Neisseria meningitidis
Authors: Swartley John S. , Liu Li-Jun , Miller Yoon K. , Martin Larry E. , Edupuganti Srilatha , Stephens David S.
Published: December 2019

The (α1→6)-linked
N
-acetyl-
d
-mannosamine-1-phosphate meningococcal capsule of serogroup A
Neisseria meningitidis
is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene,
ctrA
, and
galE
, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from
ctrA
by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to
ctrA
and that transcription of the ORFs was initiated from the intergenic region by a ς-70-type promoter that overlapped the
ctrA
promoter. The first ORF exhibited 58% amino acid identity with the UDP-
N
-acetyl-
d
-glucosamine (UDP-GlcNAc) 2-epimerase of
Escherichia coli
, which is responsible for the conversion of UDP-GlcNAc into UDP-
N
-acetyl-
d
-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A
N. meningitidis
-specific operon located between
ctrA
and
galE
.
Hybrid
Bordetella pertussis-Escherichia coli
RNA Polymerases: Selectivity of Promoter Activation
Authors: Steffen Pierre , Ullmann Agnes
Published: December 2019

We constructed hybrid
Bordetella pertussis-Escherichia coli
RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of
B. pertussis
, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the
E. coli lac
promoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the
E. coli
α subunit. This suggests that activator contact sites involve a high degree of selectivity.
Trehalose Is Not Relevant for In Vivo Activity of ς
S
-Containing RNA Polymerase in
Escherichia coli
Authors: Germer Jens , Muffler Andrea , Hengge-Aronis Regine
Published: December 2019

The ς
S
- and ς
70
-associated forms of RNA polymerase core enzyme (E) of
Escherichia coli
have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on ς
S
. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of Eς
S
and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649–3654, 1997). However, we demonstrate here that a trehalose-free
otsA
mutant exhibits growth phase-related and osmotic induction of various ς
S
-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of ς
S
-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.
An Archaeal Aerotaxis Transducer Combines Subunit I Core Structures of Eukaryotic Cytochrome
c
Oxidase and Eubacterial Methyl-Accepting Chemotaxis Proteins
Authors: Brooun Alexei , Bell James , Freitas Tracey , Larsen Randy W. , Alam Maqsudul
Published: December 2019

Signal transduction in the archaeon
Halobacterium salinarum
is mediated by three distinct subfamilies of transducer proteins. Here we report the complete
htrVIII
gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome
c
oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the
htrVIII
gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.
The Cell Wall-Anchored
Streptomyces reticuli
Avicel-Binding Protein (AbpS) and Its Gene
Authors: Walter Stefan , Wellmann Egbert , Schrempf Hildgund
Published: December 2019

Streptomyces reticuli
produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located α-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.
Roles of the Catalytic Domain and Two Cellulose Binding Domains of
Thermomonospora fusca
E4 in Cellulose Hydrolysis
Authors: Irwin Diana , Shin Dong-Hoon , Zhang Sheng , Barr Brian K. , Sakon Joshua , Karplus P. Andrew , Wilson David B.
Published: December 2019

Thermomonospora fusca
E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.
The TolQRA Proteins Are Required for Membrane Insertion of the Major Capsid Protein of the Filamentous Phage f1 during Infection
Authors: Click Eva Marie , Webster Robert E.
Published: December 2019

Infection of
Escherichia coli
by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in
tolQ
, -
R
and -
A
. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.
Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of
Nocardia corallina
B276
Authors: Allen Jeffrey R. , Ensign Scott A.
Published: December 2019

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete
Nocardia corallina
B276 was investigated. Suspensions of
N. corallina
cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of
N. corallina
was stimulated by the addition of CO
2
and inhibited by the depletion of CO
2
. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO
2
, NAD
+
, and a reductant (NADPH or dithiothreitol). In the absence of CO
2
, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in
N. corallina
occurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium
Xanthobacter
strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I from
Xanthobacter
strain Py2 to methylepoxypropane-inactivated
N. corallina
extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from
Xanthobacter
Py2 to active
N. corallina
extracts stimulated epoxide isomerase rates to the same levels observed with the purified
Xanthobacter
system. Antibodies raised against
Xanthobacter
strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown
N. corallina
extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO
2
fixation and the activity of a multicomponent epoxide carboxylase enzyme system.
Lysis Genes of the
Bacillus subtilis
Defective Prophage PBSX
Authors: Krogh Susanne , Jørgensen Steen T. , Devine Kevin M.
Published: December 2019

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are
xepA
, encoding an exported protein;
xhlA
, encoding a putative membrane-associated protein;
xhlB
, encoding a putative holin; and
xlyA
, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of
Bacillus subtilis
168. The results show that
xepA
is unlikely to be involved in host cell lysis. Expression of both
xhlA
and
xhlB
is necessary to effect host cell lysis of
B. subtilis
. Expression of
xhlB
(encoding the putative holin) together with
xlyA
(encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when
xlyA
is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.
A Membrane-Associated Protein, FliX, Is Required for an Early Step in
Caulobacter
Flagellar Assembly
Authors: Mohr Christian D. , MacKichan Joanna K. , Shapiro Lucy
Published: December 2019

The ordered assembly of the
Caulobacter crescentus
flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarachy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene,
fliX
. A
fliX
null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed
fliX
within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The
fliX
gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of
fliX
is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in
Caulobacter
predivisional cells. Full expression of
fliX
was found to be dependent on
ctrA
, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the
fliX
promoter. The
fliX
gene is located upstream and is divergently transcribed from the class III flagellar gene
flgI
, which encodes the basal body P-ring monomer. Analysis of the
fliX-flgI
intergenic region revealed an arrangement of
cis
-acting elements similar to that of another set of
Caulobacter
class II and class III flagellar genes,
fliL-flgF
, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.
The
Bacillus subtilis
DinR Binding Site: Redefinition of the Consensus Sequence
Authors: Winterling Kevin W. , Chafin David , Hayes Jeffery J. , Sun Ji , Levine Arthur S. , Yasbin Ronald E. , Woodgate Roger
Published: December 2019

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium
Bacillus subtilis
. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N
4
-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the
dinA
,
dinB
,
dinC
, and
dinR
genes. Furthermore, detailed mutational analysis of the
B. subtilis recA
operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N
4
region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the
B. subtilis dinR
and
recA
DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.
Nuclease Overexpression Mutants of
Serratia marcescens
Authors: Guynn Linda J. , Dai Wenping , Benedik Michael J.
Published: December 2019

A family of mutants overexpressing the
Serratia marcescens
extracellular nuclease has been known for decades. A number of these alleles are characterized here at the molecular level, and the mutant genes are identified, yielding a likely model for their phenotype. The known mutations exert their effect indirectly on
nucA
expression by elevating the basal SOS response of the cell. Mutations have been found in
xerC
and
uvrD
, both of which result in partial SOS induction. A classic

nuc
su

allele, that of strain W1050, is also likely to be in
xerC.
The Pol β-14 Dominant Negative Rat DNA Polymerase β Mutator Mutant Commits Errors during the Gap-Filling Step of Base Excision Repair in
Saccharomyces cerevisiae
Authors: Clairmont Caroline A. , Sweasy Joann B.
Published: December 2019

We demonstrated recently that dominant negative mutants of rat DNA polymerase β (Pol β) interfere with repair of alkylation damage in
Saccharomyces cerevisiae
. To identify the alkylation repair pathway that is disrupted by the Pol β dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol β mutants to genes known to be involved in repair of DNA alkylation damage in
S. cerevisiae
. We demonstrate that the rat Pol β mutants interfere with the base excision repair pathway in
S. cerevisiae
. In addition, expression of one of the Pol β dominant negative mutants, Pol β-14, increases the spontaneous mutation rate of
S. cerevisiae
whereas expression of another Pol β dominant negative mutant, Pol β-TR, does not. Expression of the Pol β-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol β-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.
Activation of the Proprotein Transcription Factor Pro-ς
E
Is Associated with Its Progression through Three Patterns of Subcellular Localization during Sporulation in
Bacillus subtilis
Authors: Hofmeister Antje
Published: December 2019

The activity of the sporulation transcription factor ς
E
in
Bacillus subtilis
is governed by an intercellular signal transduction pathway that controls the conversion of the inactive proprotein pro-ς
E
to the mature and active form of the factor. Here I use immunofluorescence microscopy to show that the activation of the proprotein is associated with its progression through three patterns of subcellular localization. In the predivisional sporangium, pro-ς
E
was found to be associated with the cytoplasmic membrane. Next, at the stage of asymmetric division, pro-ς
E
accumulated at the sporulation septum. Finally, after processing, mature ς
E
was found to be distributed throughout the mother cell cytoplasm. The results of subcellular fractionation and sedimentation in density gradients of extracts prepared from postdivisional sporangia confirmed that pro-ς
E
was chiefly present in the membrane fraction and that ς
E
was predominantly cytoplasmic, findings that suggest that the pro-amino acid sequence is responsible for the sequestration of pro-ς
E
to the membrane. The results of chemical cross-linking experiments showed that pro-ς
E
was present in a complex with its putative processing protein, SpoIIGA, or with a protein that depended on SpoIIGA. The membrane association of pro-ς
E
was, however, independent of SpoIIGA and other proteins specific to
B. subtilis
. Likewise, accumulation of pro-ς
E
at the septum did not depend on its interaction with SpoIIGA. Sequestration of pro-ς
E
to the membrane might serve to facilitate its interaction with SpoIIGA and may be important for preventing its premature association with core RNA polymerase. The implications of these findings for the compartmentalization of ς
E
are discussed.