bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting the VanRS Signal Transduction Pathway with Specific Inhibitors
Authors: Ulijasz Andrew T. , Weisblum Bernard 
Published: December 2019

ABSTRACT

The VanRS two-component signal transduction pathway from
Enterococcus faecium
was reconstituted in vitro from partially purified components and shown to be inhibited by the halophenyl isothiazolone LY-266,400, inhibitor A, a compound shown previously to reduce expression of the AlgR1-AlgR2 two-component signal transduction pathway in
Pseudomonas aeruginosa
(S. Roychoudhury, N. A. Zielinski, A. J. Ninfa, N. E. Allen, L. N. Jungheim, T. I. Nicas, and A. M. Chakrabarty, Proc. Natl. Acad. Sci. USA 90:965–969, 1993). Inhibitor A attenuates phosphoryl transfer from VanS∼P to VanR by its action on the ability of VanR to accept. We observed an apparent stimulatory effect of inhibitor A on VanS autophosphorylation which is attributable to the accumulation of VanS∼P as an intermediate unable to transfer P
i
to the inhibited VanR. Thus, inhibitor A acts on the second of two sequential steps which lead to transcriptional activation of the VanHAXYZ gene cluster and the resultant expression of vancomycin resistance.
Targeted Mutagenesis by Duplication Insertion in the Radioresistant Bacterium
Deinococcus radiodurans
: Radiation Sensitivities of Catalase (
katA
) and Superoxide Dismutase (
sodA
) Mutants
Authors: Markillie Lye Meng , Varnum Susan M. , Hradecky Preston , Wong Kwong-Kwok 
Published: December 2019

ABSTRACT

Deinococcus radiodurans
R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (
katA
) and superoxide dismutase (
sodA
) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.
Subunit II ofBacillus subtilisCytochromecOxidase Is a Lipoprotein
Authors: Bengtsson Jenny , Tjalsma Harold , Rivolta Carlo , Hederstedt Lars 
Published: December 2019

ABSTRACTThe sequence of the N-terminal end of the deducedctaCgene product ofBacillusspecies has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochromecaa3, which is a cytochromecoxidase. UsingBacillus subtilismutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochromecdomain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochromecaa3enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.
Analysis of Protein Synthesis Rates after Initiation of Chromosome Replication in
Escherichia coli
Authors: Bechtloff Dorothée , Grünenfelder Björn , Åkerlund Thomas , Nordström Kurt 
Published: December 2019

ABSTRACT

The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in
Escherichia coli
. An
intR1
strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated
oriC
, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins. The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the
intR1
strain. The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication. These three proteins were analyzed by peptide mass mapping and appeared to be the products of the
dps
,
gapA
, and
pyrI
genes. Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear.
Functional Analysis of Glycosyltransferase Genes from
Lactococcus lactis
and Other Gram-Positive Cocci: Complementation, Expression, and Diversity
Authors: van Kranenburg Richard , Vos Harmjan R. , van Swam Iris I. , Kleerebezem Michiel , de Vos Willem M. 
Published: December 2019

ABSTRACT

Sixteen exopolysaccharide (EPS)-producing
Lactococcus lactis
strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the
eps
genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the
eps
gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the
eps
gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene
epsD
was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This
epsD
deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from
L. lactis
or
Streptococcus thermophilus
, complementation with priming glucosyltransferase genes involved in
L. lactis
EPS and
Streptococcus pneumoniae
capsule biosynthesis could completely restore or even increase EPS production in
L. lactis
.
Sequence and Organization of pXO1, the Large
Bacillus anthracis
Plasmid Harboring the Anthrax Toxin Genes
Authors: Okinaka R. T. , Cloud K. , Hampton O. , Hoffmaster A. R. , Hill K. K. , Keim P. , Koehler T. M. , Lamke G. , Kumano S. , Mahillon J. , Manter D. , Martinez Y. , Ricke D. , Svensson ⋕ R. , Jackson P. J. 
Published: December 2019

ABSTRACT

The
Bacillus anthracis
Sterne plasmid pXO1 was sequenced by random, “shotgun” cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a “pathogenicity island,” defined by a 44.8-kb region that is bordered by inverted IS
1627
elements at each end. This region contains the three toxin genes (
cya
,
lef
, and
pagA
), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in
Bacillus
spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.
Localization of Periplasmic Redox Proteins of
Alcaligenes faecalis
by a Modified General Method for Fractionating Gram-Negative Bacteria
Authors: Zhu Zhenyu , Sun Dapeng , Davidson Victor L. 
Published: December 2019

ABSTRACT

A lysozyme-osmotic shock method is described for fractionation of
Alcaligenes faecalis
which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome
c
were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.
Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from
Matsuebacter chitosanotabidus
3001
Authors: Park Jae Kweon , Shimono Kumiko , Ochiai Nobuhisa , Shigeru Kazutaka , Kurita Masako , Ohta Yukari , Tanaka Katsunori , Matsuda Hideyuki , Kawamukai Makoto 
Published: December 2019

ABSTRACT

The extracellular chitosanase (34,000
M
r
) produced by a novel gram-negative bacterium
Matsuebacter chitosanotabidus
3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag
+
. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire
choA
gene encoding chitosanase. Sequencing of the
choA
gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in
Escherichia coli
. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.
Exponential-Phase Glycogen Recycling Is Essential for Growth of
Mycobacterium smegmatis
Authors: Belanger Aimee E. , Hatfull Graham F. 
Published: December 2019

ABSTRACT

Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of
Mycobacterium smegmatis
that contains a mutation in
glgE
, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42°C in response to high levels of glycogen accumulation. The mutation in
glgE
is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of
garA
. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in
M. smegmatis
glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of
M. smegmatis
.
Genetic and Structural Analyses of Cytoplasmic Filaments of Wild-Type
Treponema phagedenis
and a Flagellar Filament-Deficient Mutant
Authors: Izard Jacques , Samsonoff William A. , Kinoshita Mary Beth , Limberger Ronald J. 
Published: December 2019

ABSTRACT

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments,
cfpA
, was identified in various treponemal species.
Treponema phagedenis cfpA
was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from
T. denticola
,
T. vincentii
, and
T. pallidum
subsp.
pertenue
. Wild-type
T. phagedenis
and a PFF-deficient isolate were analyzed by electron microscopy to assess the structural relationship of the cytoplasmic filaments and the PFF. The number of cytoplasmic filaments per cell of
T. phagedenis
(mean, 5.7) was compared with the number of PFF at each end of the cell (mean, 4.7); the results suggest that there is no direct one-to-one correlation at the cell end. Moreover, a structural link between these structures could not be demonstrated. The cytoplasmic filaments were also analyzed by electron microscopy at different stages of cell growth; this analysis revealed that they are cleaved before or during septum formation and before the nascent formation of PFF. A PFF-deficient mutant of
T. phagedenis
possessed cytoplasmic filaments similar to those of the wild type, suggesting that intact PFF are not required for their assembly and regulation. The extensive conservation of CfpA among pathogenic spirochetes suggests an important function, and structural analysis suggests that it is unlikely that the cytoplasmic filaments and the flagellar apparatus are physically linked.
Imbalanced Base Excision Repair Increases Spontaneous Mutation and Alkylation Sensitivity in
Escherichia coli
Authors: Posnick Lauren M. , Samson Leona D. 
Published: December 2019

ABSTRACT

Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in
Escherichia coli
. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the
Saccharomyces cerevisiae
Mag1 and the
E. coli
Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized
E. coli
to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes’ effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, and alkylation sensitivity are abasic sites and secondary lesions resulting from processing abasic sites via the base excision repair pathway.
The CpxRA Signal Transduction System of
Escherichia coli
: Growth-Related Autoactivation and Control of Unanticipated Target Operons
Authors: De Wulf Peter , Kwon Ohsuk , Lin E. C. C. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, the CpxRA two-component signal transduction system senses and responds to aggregated and misfolded proteins in the bacterial envelope. We show that CpxR-P (the phosphorylated form of the cognate response regulator) activates
cpxRA
expression in conjunction with RpoS, suggesting an involvement of the Cpx system in stationary-phase survival. Engagement of the CpxRA system in functions beyond protein management is indicated by several putative targets identified after a genomic screening for the CpxR-P recognition consensus sequence. Direct negative control of the newly identified targets
motABcheAW
(specifying motility and chemotaxis) and
tsr
(encoding the serine chemoreceptor) by CpxR-P was shown by electrophoretic mobility shift analysis and Northern hybridization. The results suggest that the CpxRA system plays a core role in an extensive stress response network in which the coordination of protein turnover and energy conservation may be the unifying element.
The
Vibrio cholerae
O139 Calcutta Bacteriophage CTXφ Is Infectious and Encodes a Novel Repressor
Authors: Davis Brigid M. , Kimsey Harvey H. , Chang William , Waldor Matthew K. 
Published: December 2019

ABSTRACT

CTXφ is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (
ctxAB
), one of the principal virulence factors of
Vibrio cholerae
; consequently, nonpathogenic strains of
V. cholerae
can be converted into toxigenic strains by CTXφ infection. O139 Calcutta strains of
V. cholerae
, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX
ET
, the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX
calc
). We found that the CTX
calc
prophage gives rise to infectious virions; thus, CTX
ET
φ is no longer the only known vector for transmission of
ctxAB
. The most functionally significant differences between the nucleotide sequences of CTX
calc
φ and CTX
ET
φ are located within the phages’ repressor genes (
rstR
calc
and
rstR
ET
, respectively) and their RstR operators. RstR
calc
is a novel, allele-specific repressor that regulates replication of CTX
calc
φ by inhibiting the activity of the
rstA
calc
promoter. RstR
calc
has no inhibitory effect upon the classical and El Tor
rstA
promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR
calc
renders a CTX
calc
lysogen immune to superinfection by CTX
calc
φ but susceptible (heteroimmune) to infection by CTX
ET
φ. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic
V. cholerae
O139 Calcutta probably arose via infection of an O139 CTX
ET
φ lysogen by CTX
calc
φ.
Mutations in the Regulatory Gene
hrpG
of
Xanthomonas campestris
pv. vesicatoria Result in Constitutive Expression of All
hrp
Genes
Authors: Wengelnik Kai , Rossier Ombeline , Bonas Ulla 
Published: December 2019

ABSTRACT

hrpG
is a key regulatory gene for transcriptional activation of pathogenicity genes (
hrp
) of
Xanthomonas campestris
pv. vesicatoria. We identified three mutations in
hrpG
which render
hrp
gene expression constitutive in normally suppressing medium. The mutations in
hrpG
result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutated
hrpG
enhances the timing and intensity of plant reactions in infection assays.
The BldD Protein from
Streptomyces coelicolor
Is a DNA-Binding Protein
Authors: Elliot Marie A. , Leskiw Brenda K. 
Published: December 2019

ABSTRACT

Gel mobility shift assays with His-tagged BldD isolated from
Escherichia coli
have illustrated that BldD is capable of specifically recognizing its own promoter region. DNase I and hydroxyl radical footprinting assays have served to delimit the BldD binding site, revealing that BldD recognizes and binds to a site just upstream from, and overlapping with, the −10 region of the promoter. How BldD binds to its promoter and the effect this binding has on the expression of BldD are discussed.
The IntI1 Integron Integrase Preferentially Binds Single-Stranded DNA of the
attC
Site
Authors: Francia M. Victoria , Zabala Juan C. , de la Cruz Fernando , García Lobo Juan M. 
Published: December 2019

ABSTRACT

IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites
attI
and
aadA1 attC
. When using double-stranded DNA as a substrate, we observed IntI1 binding to
attI
but not to
attC
. IntI1-COOH did not bind either
attI
or
attC
, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded
attI
. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss
attC
DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss
attC
as well as did the complete integrase, indicating that the ability of the protein to bind ss
aadA1 attC
was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss
attI
DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the
attI
bottom strand, indicating similar capabilities of IntI1 for binding
attI
DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of
aadA1 attC
against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.
Thickness and Elasticity of Gram-Negative Murein Sacculi Measured by Atomic Force Microscopy
Authors: Yao X. , Jericho M. , Pink D. , Beveridge T. 
Published: December 2019

ABSTRACTAtomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi fromEscherichia coliK-12 andPseudomonas aeruginosaPAO1. Air-dried sacculi fromE. colihad a thickness of 3.0 nm, whereas those fromP. aeruginosawere 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. WhenE. colisacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 × 107N/m2was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 × 108to 4 × 108N/m2and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intactE. colisacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927–953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301–366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.