bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Expression of the β-Glucoside (
bgl
) Operon of
Escherichia coli
Occurs in Mouse Liver
Authors: Khan M. Ayub , Isaacson Richard E. 
Published: December 2019

ABSTRACT

An
Escherichia coli
DNA fragment was identified that contained part of the β-glucoside (
bgl
) operon. This fragment was identified because it contained a promoter that was responsible for the expression of a reporter gene, the chloramphenicol acetyltransferase gene, in a mouse liver during bacterial infection but not when a bacterial clone was grown in vitro. This fragment contained a promoter and a rho-independent transcription terminator which were flanked by the 3′ end of
bglG
and the 5′ end of
bglF
. Reverse transcription-PCR confirmed that
cat
-specific mRNA was produced in infected mouse liver but not in vitro. mRNA encoding the positive regulator of the
bgl
operon,
bglG
, also was detected in mouse liver infected with an
E. coli
strain. These results demonstrated that expression of the
bgl
operon occurs in infected mouse liver and suggests a unique role for this operon in vivo.
The
pcd
Gene Encoding Piperideine-6-Carboxylate Dehydrogenase Involved in Biosynthesis of α-Aminoadipic Acid Is Located in the Cephamycin Cluster of
Streptomyces clavuligerus
Authors: Pérez-Llarena Francisco J. , Rodríguez-García Antonio , Enguita Francisco J. , Martín Juan F. , Liras Paloma 
Published: December 2019

ABSTRACT

Three open reading frames (ORFs) have been located downstream of
cefE
in the cephamycin C gene cluster of
Streptomyces clavuligerus
. ORF13 (
pcd
) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (
cmcT
) encodes a 523-amino-acid protein (MW, 54,232) analogous to
Streptomyces
proteins for efflux and resistance to antibiotics. ORF15 (
pbp74
) encodes a high molecular weight penicillin-binding protein (MW, 74,094).
Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in
Escherichia coli
K-12 Is Unrelated to the Function of the Transport System
Authors: Celis Roberto T. F. , Leadlay Peter F. , Roy Ipsita , Hansen Anne 
Published: December 2019

ABSTRACT

In
Escherichia coli
K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (
argK
) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in
E. coli
K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.
Transformation of the Lyme Disease Spirochete
Borrelia burgdorferi
with Heterologous DNA
Authors: Stevenson Brian , Bono James L. , Elias Abdallah , Tilly Kit , Rosa Patricia 
Published: December 2019

ABSTRACT

Studies of the spirochete
Borrelia burgdorferi
have been hindered by the scarcity of genetic tools that can be used in these bacteria. For the first time, a method has been developed by which heterologous DNA (DNA without a naturally occurring
B. burgdorferi
homolog) can be introduced into and persistently maintained by
B. burgdorferi
. This technique uses integration of circular DNA into the bacterial genome via a single-crossover event. The ability to transform
B. burgdorferi
with heterologous DNA will now permit a wide range of experiments on the biology of these bacteria and their involvement in the many facets of Lyme disease.
Mutational Analysis of the Transcriptional Regulator GcvA: Amino Acids Important for Activation, Repression, and DNA Binding
Authors: Jourdan Alissa D. , Stauffer George V. 
Published: December 2019

ABSTRACT

The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the
gcvTHP
operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in
gcvA
to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a
gcvT-lacZ
fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a
gcvT-lacZ
fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.
Efficient Transfer of the Pheromone-Independent
Enterococcus faecium
Plasmid pMG1 (Gm
r
) (65.1 Kilobases) to
Enterococcus
Strains during Broth Mating
Authors: Ike Yasuyoshi , Tanimoto Koichi , Tomita Haruyoshi , Takeuchi Kunio , Fujimoto Shuhei 
Published: December 2019

ABSTRACT

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant
Enterococcus faecium
clinical isolate and was found to encode gentamicin resistance.
Eco
RI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to
Enterococcus
strains in broth mating. pMG1 transferred between
Enterococcus faecalis
strains, between
E. faecium
strains, and between
E. faecium
and
E. faecalis
strains at a frequency of approximately 10
−4
per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free
E. faecalis
FA2-2 or an
E. faecium
strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of
E. faecalis
FA2-2 or an
E. faecium
strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of
Enterococcus
and that this system is different from the pheromone-induced transfer system of
E. faecalis
plasmids.
Genomic Analysis Reveals Chromosomal Variation in Natural Populations of the Uncultured Psychrophilic Archaeon
Cenarchaeum symbiosum
Authors: Schleper Christa , DeLong Edward F. , Preston Christina M. , Feldman Robert A. , Wu Ke-Ying , Swanson Ronald V. 
Published: December 2019

ABSTRACT

Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote
Cenarchaeum symbiosum
found living in association with a marine sponge,
Axinella mexicana
. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from
C. symbiosum
. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.
Formation of Single-Stranded DNA during DNA Transformation of
Neisseria gonorrhoeae
Authors: Chaussee Michael S. , Hill Stuart A. 
Published: December 2019

ABSTRACT

Neisseria gonorrhoeae
is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of
N. gonorrhoeae
. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the
dud-1
mutation. Surprisingly, ssDNA was also found during transformation of
N. gonorrhoeae comA
mutants, which suggested that ssDNA was initially formed within the periplasm.
Structural Organization of Virulence-Associated Plasmids of
Yersinia pestis
Authors: Hu Ping , Elliott Jeffrey , McCready Paula , Skowronski Evan , Garnes Jeffrey , Kobayashi Arthur , Brubaker Robert R. , Garcia Emilio 
Published: December 2019

ABSTRACT

The complete nucleotide sequence and gene organization of the three virulence plasmids from
Yersinia pestis
KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS
100
. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two
Yersinia
species pathogenic for humans (
Y. pseudotuberculosis
and
Y. enterocolitica
). A new pseudogene, which occurs as an intact gene in the
Y. enterocolitica
and
Y. pseudotuberculosis
-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in
Y. pseudotuberculosis
and
Y. enterocolitica
, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS
100
, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS
100
inserts is a partial copy of IS
285
. A single copy of an IS
200
-like element (recently named IS
1541
) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.
Genetic Analysis of Mecillinam-Resistant Mutants of
Caulobacter crescentus
Deficient in Stalk Biosynthesis
Authors: Seitz Lisa C. , Brun Yves V. 
Published: December 2019

ABSTRACT

Stalk synthesis in
Caulobacter crescentus
is a developmentally controlled and spatially restricted event that requires the synthesis of peptidoglycan at the stalk-cell body junction. We show that the β-lactam antibiotic mecillinam prevents stalk synthesis by inhibiting stalk elongation. In addition, mecillinam causes an increase in the diameter of the stalk at the stalk-cell body junction. We describe two mutations that confer resistance to mecillinam and that prevent stalk elongation. These mutations are probably allelic, and they map to a locus previously not associated with stalk synthesis.
Growth Medium-Dependent Regulation of
Myxococcus xanthus
Fatty Acid Content Is Controlled by the
esg
Locus
Authors: Bartholomeusz Geoffrey , Zhu Yanglong , Downard John 
Published: December 2019

ABSTRACT

We compared the cellular fatty acid profiles of
Myxococcus xanthus
cells grown in either a Casitone-based complex medium or a chemically defined medium. The cells grown in the complex medium had a much higher content of the abundant branched-chain fatty acid
iso
-15:0 and several other branched-chain species. The higher branched-chain fatty acid content of the cells grown in the complex medium was dependent on the
esg
locus, which encodes the E1α and E1β components of a branched-chain keto acid dehydrogenase (BCKAD) multienzyme complex involved in branched-chain fatty acid biosynthesis. Cells grown in the complex medium were also found to have a higher level of
esg
transcription and more BCKAD enzyme activity than cells from the chemically defined medium. The level of
esg
transcription appears to be an important factor in the growth medium-dependent regulation of the
M. xanthus
branched-chain fatty acid content.
Functional Analysis of the Two-Gene Lysis System of the Pneumococcal Phage Cp-1 in Homologous and Heterologous Host Cells
Authors: Martín Ana C. , López Rubens , García Pedro 
Published: December 2019

ABSTRACT

The two lysis genes
cph1
and
cpl1
of the
Streptococcus pneumoniae
bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in
Escherichia coli
. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The
cph1
gene was able to complement a lambda
S
am mutation in the nonsuppressing
E. coli
HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in
E. coli
resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the
cph1
and
cpl1
genes cloned in a pneumococcal mutant with a complete deletion of the
lytA
gene, which codes for the
S. pneumoniae
main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.
Genetic Organization of the
mecA
Region in Methicillin-Susceptible and Methicillin-Resistant Strains of
Staphylococcus sciuri
Authors: Wu Shangwei , de Lencastre Herminia , Tomasz Alexander 
Published: December 2019

ABSTRACT

A homolog of the
Staphylococcus aureus
methicillin resistance gene
mecA
was recently shown to be ubiquitous in independent isolates of the animal species
Staphylococcus sciuri
. The
mecA
gene homolog and regions flanking it were cloned and sequenced from four strains of
S. sciuri
: strain K1 (ATCC 29062), a representative of
S. sciuri
subsp.
sciuri
; two strains (K3 and K8) representing
S. sciuri
subsp.
rodentius
; and strain K11, a representative of
S. sciuri
subsp.
carnaticum
. Strains K1 and K11 were susceptible to methicillin, while strains K3 and K8 showed heterogeneous resistance. The
mecA
genes of strains K1 and K11 and one of the two copies of
mecA
(
mecA1
) present in strain K3 had virtually identical DNA sequences in the
mecA
gene and were similar in genetic organization in the flanking regions. In contrast, the single copy of
mecA
in strain K8 and the second copy of
mecA
(
mecA2
) in strain K3 had
mecA
DNA sequences identical to that of
S. aureus mecA
, and the
mecA
region in these two strains was also similar to that of the same region in the
S. aureus
strain used for comparison. Interestingly, an open reading frame defining an N-terminal truncated polypeptide, NTORF101, with a high degree of homology to a DNA segment in the hypervariable region of methicillin-resistant
S. aureus
(and also similar to the
Escherichia coli
gene
ugpQ
) was also identified downstream of the
mecA
homolog of strain K11, representing
S. sciuri
subsp.
carnaticum
. The
ugpQ
-like gene is not present in methicillin-susceptible strains of
S. aureus
. The presence of such a
ugpQ
-like gene together with the homolog of
mecA
in strain K11 supports the speculation that these genetic elements may be evolutionary relatives and/or precursors of the genetic determinant of methicillin resistance in
S. aureus.
Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolylcis-transIsomerase from a Thermophilic Archaeon,Methanococcus thermolithotrophicus
Authors: Furutani Masahiro , Iida Toshii , Yamano Shigeyuki , Kamino Kei , Maruyama Tadashi 
Published: December 2019

ABSTRACTA peptidyl prolylcis-transisomerase (PPIase) was purified from a thermophilic methanogen,Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (kcat/Km) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicusgenomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.
Effects of Abnormal-Sterol Accumulation on
Ustilago maydis
Plasma Membrane H
+
-ATPase Stoichiometry and Polypeptide Pattern
Authors: Hernández Agustín , Cooke David T. , Clarkson David T. 
Published: December 2019

ABSTRACT

Accumulation of 14α-methylated sterols or Δ
8
-sterols in
Ustilago maydis
affected three aspects of the plasma membrane H
+
-ATPase. Proton transport was reduced in Δ
8
-sterol-accumulating samples, due to an altered H
+
/ATP stoichiometry. ATP hydrolytic activity was increased, but no direct correlation with the extent or type of abnormal sterol accumulated could be drawn. Finally, Western blot analysis with antibodies against yeast PMA1 revealed a second lighter band (99-kDa band) in all samples from abnormal-sterol-accumulating sporidia. The conclusions are that the 99-kDa band and a reduced stoichiometry are directly linked to the presence of abnormal sterols, while changes in hydrolytic activity are linked only indirectly.
Role of
Agrobacterium virB
Genes in Transfer of T Complexes and RSF1010
Authors: Fullner Karla Jean 
Published: December 2019

ABSTRACT

Nonpolar
virB
mutants of
Agrobacterium tumefaciens
were tested for RSF1010 mobilization and extracellular complementation.
virB2
to
virB11
were essential for transfer in both assays.
virB1
was essential only for high frequency transfer of RSF1010 and VirE2. Coordinated transfer of a preassembled T complex is supported by these data and competition studies.
The Transfer Origin for
Bacteroides
Mobilizable Transposon Tn
4555
Is Related to a Plasmid Family from Gram-Positive Bacteria
Authors: Smith C. Jeffrey , Parker Anita C. 
Published: December 2019

ABSTRACT

Conjugal transfer of
Bacteroides
mobilizable transposon Tn
4555
was examined with an
Escherichia coli
-based assay system. It was shown that mobilization required the
cis
-acting
oriT
Tn
region and that the Tn
4555 mobA
Tn
gene and RK231 must be present in
trans
. With alkaline agarose gel electrophoresis and filter blot hybridizations, it was shown that at
oriT
Tn
there was a site- and strand-specific cleavage event that was dependent on
mobA
Tn
. The 5′ end of this cleavage site was mapped by primer extension, and the nucleotide sequence surrounding the site had homology to a family of
oriT
nick sites found in mobilizable plasmids of gram-positive bacteria. Removal of the nick site by deletion of 18 bp surrounding the site resulted in a significant loss of transfer activity.
A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in
Myxococcus xanthus
Authors: Weimer Robby M. , Creighton Chad , Stassinopoulos Angela , Youderian Philip , Hartzell Patricia L. 
Published: December 2019

ABSTRACT

Three independent Tn
5-lac
insertions in the S1 locus of
Myxococcus xanthus
inactivate the
sglK
gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of
sglK
reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The
sglK
gene is cotranscribed with the upstream
grpS
gene, which encodes a GrpE homologue. Unlike
sglK
,
grpS
is not required for social motility or development. Wild-type
M. xanthus
is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in
sglK
inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that
sglK
mutants do not make fibrils. The
fibR
gene, located immediately upstream of the
grpS-sglK
operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in
Pseudomonas aeruginosa
and
Pseudomonas putida
. Inactivation of
fibR
leads to the overproduction of fibrillin, suggesting that
M. xanthus
fibril production and
Pseudomonas
alginate production are regulated in analogous ways.
M. xanthus
and
Pseudomonas
exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.