bluuebunny/crossref_metadata_2025 huggingface dataset:
Farnesol-Induced Generation of Reactive Oxygen Species via Indirect Inhibition of the Mitochondrial Electron Transport Chain in the Yeast
Saccharomyces cerevisiae
Authors: Machida Kiyotaka , Tanaka Toshio , Fujita Ken-ichi , Taniguchi Makoto
Published: December 2019

The mechanism of farnesol (FOH)-induced growth inhibition of
Saccharomyces cerevisiae
was studied in terms of its promotive effect on generation of reactive oxygen species (ROS). The level of ROS generation in FOH-treated cells increased five- to eightfold upon the initial 30-min incubation, while cells treated with other isoprenoid compounds, like geraniol, geranylgeraniol, and squalene, showed no ROS-generating response. The dependence of FOH-induced growth inhibition on such an oxidative stress was confirmed by the protection against such growth inhibition in the presence of an antioxidant such as α-tocopherol, probucol, or
N
-acetylcysteine. FOH could accelerate ROS generation only in cells of the wild-type grande strain, not in those of the respiration-deficient petite mutant ([
rho
0
]), which illustrates the role of the mitochondrial electron transport chain as its origin. Among the respiratory chain inhibitors, ROS generation could be effectively eliminated with myxothiazol, which inhibits oxidation of ubiquinol to the ubisemiquinone radical by the Rieske iron-sulfur center of complex III, but not with antimycin A, an inhibitor of electron transport that is functional in further oxidation of the ubisemiquinone radical to ubiquinone in the Q cycle of complex III. Cellular oxygen consumption was inhibited immediately upon extracellular addition of FOH, whereas FOH and its possible metabolites failed to directly inhibit any oxidase activities detected with the isolated mitochondrial preparation. A protein kinase C (PKC)-dependent mechanism was suggested to exist in the inhibition of mitochondrial electron transport since FOH-induced ROS generation could be effectively eliminated with a membrane-permeable diacylglycerol analog which can activate PKC. The present study supports the idea that FOH inhibits the ability of the electron transport chain to accelerate ROS production via interference with a phosphatidylinositol type of signal.
Transcription Analysis of the Prolate-Headed Lactococcal Bacteriophage c2
Authors: Lubbers Mark W. , Schofield Karin , Waterfield Nick R. , Polzin Kayla M.
Published: December 2019

A detailed transcription map of the prolate-headed lactococcal phage c2 has been constructed. Transcription of about one-third of the genome, encoding 22 open reading frames, began within the first 2 min of infection and produced at least 12 overlapping transcripts that persisted until lysis occurred at 30 min after initiation of infection. The remaining two-thirds of the genome, encoding 17 open reading frames, was divergently transcribed, beginning between 4 and 6 min after initiation of infection, and resulted in at least 18 overlapping transcripts that persisted until lysis. Five very strong, simultaneously active, and probably unregulated early promoters and a single positively regulated late promoter were identified. The late promoter had an extended −10 sequence, had a significant basal level of activity in the uninduced state, and was induced to high activity by a phage gene product. The complex overlapping pattern of transcripts resulted from the action of the multiple early promoters, inefficient termination of transcription, and (possibly) processing of a late precursor transcript(s). Phage proteins were not required for these processes, and the host RNA polymerase was probably used for both early and late transcription.
Identification of the Gene Encoding the Alternative Sigma Factor ς
B
from
Listeria monocytogenes
and Its Role in Osmotolerance
Authors: Becker Lynne A. , Çetin Mehmet Sevket , Hutkins Robert W. , Benson Andrew K.
Published: December 2019

Listeria monocytogenes
is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by
L. monocytogenes
to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the
sigB
gene and several
rsb
genes from
L. monocytogenes
, encoding homologs of the alternative sigma factor ς
B
and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium
Bacillus subtilis
. The
L. monocytogenes
and
B. subtilis sigB
and
rsb
genes are similar in sequence and physical organization; however, we observed that the activity of ς
B
in
L. monocytogenes
was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for ς
B
in coordinating osmotic responses in
L. monocytogenes
. A null mutation in the
sigB
gene led to substantial defects in the ability of
L. monocytogenes
to use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of ς
B
reduced the ability of the cells to accumulate betaine. Thus, ς
B
coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of
L. monocytogenes
under conditions of high osmotic strength.
Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in
Escherichia coli
Authors: Subrahmanyam Satyanarayana , Cronan John E.
Published: December 2019

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three
Escherichia coli
isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to
E. coli
, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.
Physiological Response of
Lactobacillus plantarum
to Salt and Nonelectrolyte Stress
Authors: Glaasker Erwin , Tjan Frans S. B. , Ter Steeg Pieter F. , Konings Wil N. , Poolman Bert
Published: December 2019

In this report, we compared the effects on the growth of
Lactobacillus plantarum
of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K
+
(or Na
+
) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.
Salmonella typhimurium nit
Is
nadE
: Defective Nitrogen Utilization and Ammonia-Dependent NAD Synthetase
Authors: Schneider Barbara L. , Reitzer Lawrence J.
Published: December 2019

S. typhimurium nit
mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes. Complementation, enzyme assays, and genetic mapping show that
nit
is
nadE
. We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

A 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

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

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

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

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.
Autoregulation of the pTF-FC2 Proteic Poison-Antidote Plasmid Addiction System (
pas
) Is Essential for Plasmid Stabilization
Authors: Smith Anthony S. G. , Rawlings Douglas E.
Published: December 2019

The
pasABC
genes of the proteic plasmid addiction system of broad-host-range plasmid pTF-FC2 were autoregulated. The PasA antidote was able to repress the operon 25-fold on its own, and repression was increased to 100-fold when the PasB toxin was also present. Autoregulation appears to be an essential requirement for
pas
-mediated plasmid stabilization because when the
pas
genes were placed behind the isopropyl-β-
d
-thiogalactopyranoside (IPTG)-regulated
tac
promoter, they were unable to stabilize a heterologous test plasmid.
Int-B13, an Unusual Site-Specific Recombinase of the Bacteriophage P4 Integrase Family, Is Responsible for Chromosomal Insertion of the 105-Kilobase
clc
Element of
Pseudomonas
sp. Strain B13
Authors: Ravatn Roald , Studer Sonja , Zehnder Alexander J. B. , Roelof van der Meer Jan
Published: December 2019

Pseudomonas
sp. strain B13 carries the
clcRABDE
genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb
clc
element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (
glyV
) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (
int-B13
) was located near the right end of the
clc
element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in
Pseudomonas aeruginosa
PaK1. Similar to the bacteriophages φR73 and P4, the
clc
element integrates into the 3′ end of the target tRNA gene. This target site was characterized from four different recipient strains into which the
clc
element integrated, showing sequence specificity of the integration. In
Pseudomonas
sp. strain B13, a circular form of the
clc
element, which carries an 18-bp DNA sequence identical to the 3′-end portion of
glyV
as part of its attachment site (
attP
), could be detected. Upon chromosomal integration of the
clc
element into a bacterial attachment site (
attB
), a functional
glyV
was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient
Escherichia coli
with two recombinant plasmids, one carrying the
int-B13
gene and the
attP
site and the other carrying the
attB
site of
Pseudomonas putida
F1.
Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in
Pseudomonas aeruginosa
Authors: Nishijyo Takayuki , Park Seung-Moon , Lu Chung-Dar , Itoh Yoshifumi , Abdelal Ahmed T.
Published: December 2019

The complete nucleotide sequence for the
aot
operon of
Pseudomonas aeruginosa
PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these,
aotJ
,
aotQ
,
aotM
, and
aotP
, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The
aotO
gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an
aotJ
::
lacZ
translational fusion showed that expression of the
aot
operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible
aru
promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in
P. aeruginosa
. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.
Wound-Released Chemical Signals May Elicit Multiple Responses from an
Agrobacterium tumefaciens
Strain Containing an Octopine-Type Ti Plasmid
Authors: Kalogeraki Virginia S. , Winans Stephen C.
Published: December 2019

The
vir
regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from
Agrobacterium tumefaciens
to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two
vir
regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb
virH
locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The
virH
operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated
virK
. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the
vir
regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to
lacZ
. Expression of these genes was also measured by nuclease S1 protection assays.
virK
and two nonconserved genes, designated
virL
and
virM
, were strongly induced by the
vir
gene inducer acetosyringone. Disruptions of
virH
,
virK
,
virL
, or
virM
did not affect tumorigenesis of
Kalanchöe diagramontiana
leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.
Deletion of the 389 N-Terminal Residues of the Transcriptional Activator AREA Does Not Result in Nitrogen Metabolite Derepression in
Aspergillus nidulans
Authors: Caddick Mark X. , Arst Herbert N.
Published: December 2019

Utilizing a homologous gene replacement in order to retain the native promoter and 5′ and 3′ untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression in
Aspergillus nidulans
. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649–6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.
Transcriptional Regulation of the
Streptococcus salivarius
57.I Urease Operon
Authors: Chen Yi-Ywan M. , Weaver Cheryl A. , Mendelsohn David R. , Burne Robert A.
Published: December 2019

The
Streptococcus salivarius
57.I
ure
cluster was organized as an operon, beginning with
ureI
, followed by
ureABC
(structural genes) and
ureEFGD
(accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A ς
70
-like promoter could be mapped 5′ to
ureI
(
PureI
) by primer extension analysis. The intensity of the signal increased when cells were grown at an acidic pH and was further enhanced by excess carbohydrate. To determine the function(s) of two inverted repeats located 5′ to
PureI
, transcriptional fusions of the full-length promoter region (
PureI
), or a deletion derivative (
PureI
Δ100), and a promoterless chloramphenicol acetyltransferase (CAT) gene were constructed and integrated into the chromosome to generate strains
PureI
CAT and
PureI
Δ100CAT, respectively. CAT specific activities of
PureI
CAT were repressed at pH 7.0 and induced at pH 5.5 and by excess carbohydrate. In
PureI
Δ100CAT, CAT activity was 60-fold higher than in
PureI
CAT at pH 7.0 and pH induction was nearly eliminated, indicating that expression was negatively regulated. Thus, it was concluded that
PureI
was the predominant, regulated promoter and that regulation was governed by a mechanism differing markedly from other known mechanisms for bacterial urease expression.
trans
-Complementation of a
Staphylococcus aureus agr
Mutant by
Staphylococcus lugdunensis agr
RNAIII
Authors: Benito Yvonne , Lina Gerard , Greenland Timothy , Etienne Jerome , Vandenesch François
Published: December 2019

RNAIII from
Staphylococcus lugdunensis
(RNAIII-sl) in a
Staphylococcus aureus agr
mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5′ end of RNAIII-sl and the 3′ end of RNAIII from
S. aureus
restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.
Molecular Characterization of the
Lactococcus lactis Lla
KR2I Restriction-Modification System and Effect of an IS
982
Element Positioned between the Restriction and Modification Genes
Authors: Twomey Denis P. , McKay Larry L. , O’Sullivan Daniel J.
Published: December 2019

The nucleotide sequence of the plasmid-encoded
Lla
KR2I restriction-modification (R-M) system of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (
llaKR2IR
) and methyltransferase (
llaKR2IM
) genes; located in the intergenic region is a copy of the insertion element IS
982
, whose putative transposase gene is codirectionally transcribed with
llaKR2IM
. The deduced sequence of the
Lla
KR2I endonuclease shared homology with the type II endonuclease
Sau
3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5′ GATC 3′. In addition, M · 
Lla
KR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M · 
Sau
3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M · 
Lla
KR2I, all remained undigested after treatment with
Sau
3AI despite the presence of multiple 5′ GATC 3′ sites. The combination of these data suggested that the specificity of the
Lla
KR2I R-M system was likely to be 5′ GATC 3′, with the cytosine residue being modified to 5-methylcytosine. The IS
982
element located within the
Lla
KR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the
llaKR2IR
gene, was shown to overlap the direct repeat sequence on the other side of IS
982
. Specific deletion of IS
982
and one of these direct repeats via a PCR strategy indicated that the
Lla
KR2I R-M determinants do not rely on elements within IS
982
for expression and that the efficiency of bacteriophage restriction was not impaired.
Transcriptional Activation of the
Bacillus subtilis ackA
Gene Requires Sequences Upstream of the Promoter
Authors: Turinsky Andrew J. , Grundy Frank J. , Kim Jeong-Ho , Chambliss Glenn H. , Henkin Tina M.
Published: December 2019

Transcriptional activation of the
Bacillus subtilis ackA
gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the
ackA
promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in
B. subtilis
and other gram-positive bacteria, recognizes a
cis
-acting consensus sequence, designated
cre
(catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions −116.5 and −56.5 of the
ackA
promoter and have been termed
cre1
and
cre2
, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study,
cre2
, the site closer to the promoter, and the region upstream of
cre2
were shown to be indispensable for CcpA-dependent transcriptional activation of
ackA
, whereas
cre1
was not required. In addition, insertion of 5 bp between
cre2
and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of
cre2
and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro to
cre2
but not
cre1
, consistent with the genetic data. Activation of
ackA
transcription was blocked in a
ptsH1/crh
double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.
The Carboxyl Terminus of Protein TraD Adds Specificity and Efficiency to F-Plasmid Conjugative Transfer
Authors: Sastre J. Ignacio , Cabezón Elena , de la Cruz Fernando
Published: December 2019

We isolated and characterized
traD
mutants with an altered specificity of interaction with relaxosomes of various conjugative (F and R388) and mobilizable (RSF1010 and ColE1) plasmids. The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer.
Cyanobacterial Transposons Tn
5469
and Tn
5541
Represent a Novel Noncomposite Transposon Family
Authors: Schaefer Michael R. , Kahn Katherine
Published: December 2019

A noncomposite transposon, designated Tn
5541
, was isolated from strain Fd33 of the filamentous cyanobacterium
Fremyella diplosiphon
UTEX 481. Sequence analysis showed that Tn
5541
is structurally and genetically very similar to Tn
5469
, which is also endogenous to
F. diplosiphon
. Both Tn
5469
and Tn
5541
encode homologous forms of an unusual composite transposase and a protein of unknown function. DNA hybridization analysis showed that like Tn
5469
, Tn
5541
was not widely distributed among cyanobacterial genera. A similar analysis showed that Tn
5469
and Tn
5541
were equally limited to and present as multiple genomic copies in three of six distinct strains comprising the
Tolypothrix
1 cluster of heterocyst-forming filamentous cyanobacteria. These and other distinguishing features suggest that Tn
5469
and Tn
5541
represent a novel noncomposite transposon family.