bluuebunny/crossref_metadata_2025 huggingface dataset:
The
bspA
Locus of
Lactobacillus fermentum
BR11 Encodes an
l
-Cystine Uptake System
Authors: Turner Mark S. , Woodberry Tonia , Hafner Louise M. , Giffard Philip M.
Published: December 2019

BspA is a basic surface-exposed protein from
Lactobacillus fermentum
BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from
Lactobacillus reuteri
. Compared with the database of
Escherichia coli
proteins, BspA is most similar to the
l
-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up
l
-cystine. Uptake rates of
l
-glutamine,
l
-histidine, and
l
-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of
L. fermentum
BR11 with
l
-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed.
L. fermentum
BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.
A Conserved Domain in
Escherichia coli
Lon Protease Is Involved in Substrate Discriminator Activity
Authors: Ebel Wolfgang , Skinner Monica M. , Dierksen Karen P. , Scott Janelle M. , Trempy Janine E.
Published: December 2019

Lon protease of
Escherichia coli
regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Little is known about the mechanism of substrate recognition by Lon. To examine the interaction of Lon with two of its substrates, RcsA and SulA, we generated point mutations in
lon
which affected its substrate specificity. The most informative
lon
mutant overproduced capsular polysaccharide (RcsA stabilized) yet was resistant to DNA-damaging agents (SulA degraded). Immunoblots revealed that RcsA protein persisted in this mutant whereas SulA protein was rapidly degraded. The mutant contains a single-base change within
lon
leading to a single amino acid change of glutamate 240 to lysine. E240 is conserved among all Lon isolates and resides in a charged domain that has a high probability of adopting a coiled-coil conformation. This conformation, implicated in mediating protein-protein interactions, appears to confer substrate discriminator activity on Lon. We propose a model suggesting that this coiled-coil domain represents the discriminator site of Lon.
Replacement of Vegetative ς
A
by Sporulation-Specific ς
F
as a Component of the RNA Polymerase Holoenzyme in Sporulating
Bacillus subtilis
Authors: Lord Matthew , Barillà Daniela , Yudkin Michael D.
Published: December 2019

Soon after asymmetric septation in sporulating
Bacillus subtilis
cells, ς
F
is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, ς
F
must compete with ς
A
, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for ς
A
and ς
F
, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for ς
F
was found to be about 25-fold lower than that for ς
A
. We used quantitative Western blotting to estimate the concentrations of E, ς
A
, and ς
F
in sporulating cells. The cellular concentrations of E and ς
A
were both about 7.5 μM, and neither changed significantly during the first 3 h of sporulation. The concentration of ς
F
was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of ς
F
was some twofold higher than that of ς
A
. This difference in concentration cannot adequately account for the replacement of ς
A
holoenzyme by ς
F
holoenzyme in the prespore, and it seems that some further mechanism—perhaps the synthesis or activation of an anti-ς
A
factor—must be responsible for this replacement.
The Steady-State Internal Redox State (NADH/NAD) Reflects the External Redox State and Is Correlated with Catabolic Adaptation in
Escherichia coli
Authors: de Graef Mark R. , Alexeeva Svetlana , Snoep Jacky L. , Teixeira de Mattos M. Joost
Published: December 2019

Escherichia coli
MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT ≤ 1%). A simultaneous flux from pyruvate through both PFL and the pyruvate dehydrogenase complex (PDHc) was observed. In anaerobic cultures with fumarate or nitrate as an electron acceptor, a significant flux through the PDHc was calculated on the basis of the redox balance, the measured products, and the known biochemistry. This result calls into question the common assumption that the complex cannot be active under these conditions. In vitro activity measurements of PDHc showed that the cellular content of the enzyme varied with the internal redox state and revealed an activity for dissolved oxygen tension of below 1%. Whereas Western blots showed that the E3 subunit of PDHc (dihydrolipoamide dehydrogenase) did not vary to a large extent under the conditions tested, the E2 subunit (dihydrolipoamide acetyltransferase) amount followed the trend that was found for the in vitro PDHc activity. From this it is concluded that regulation of the PDHc is exerted at the E1/E2 operon (
aceEF
). We propose that the external redox state (measured as the midpoint potentials of those terminal acceptors with which the cell has sufficient capacity to react) is reflected by the internal redox state. The latter may subsequently govern both the expression and the activity of the two pyruvate-catabolizing enzymes.
Transcriptional Organization of the
czc
Heavy-Metal Homeostasis Determinant from
Alcaligenes eutrophus
Authors: Große Cornelia , Grass Gregor , Anton Andreas , Franke Sylvia , Santos Alexander Navarrete , Lawley Blair , Brown Nigel L. , Nies Dietrich H.
Published: December 2019

The Czc system of
Alcaligenes eutrophus
mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB
2
A cation-proton antiporter. DNA sequencing of the region upstream of the
czcNICBADRS
determinant located on megaplasmid pMOL30 revealed the 5′ end of
czcN
and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of
czc
. Additional open reading frames upstream of
czc
had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide
czcN
message and a 400-nucleotide
czcI
message were found, and the presence of 6,200-nucleotide
czcCBA
message (D. Van der Lelie et al., Mol. Microbiol. 23:493–503, 1997) was confirmed. Induction of
czcN
,
czcI
,
czcCBA
, and
czcDRS
followed a similar pattern: transcription was induced best by 300 μM zinc, less by 300 μM cobalt, and only slightly by 300 μM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from
czcN
to
czcC
and from
czcD
to
czcS
, but not between
czcA
and
czcD
nor between
czcS
and a 131-amino-acid open reading frame following
czcS
. The CzcR putative response regulator was purified and shown to bind in the 5′ region of
czcN
. A reporter strain carrying a
czcNIC-lacZ-czcBADRS
determinant on plasmid pMOL30 was constructed, as were Δ
czcR
and Δ
czcS
mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn
2+
, (ii) induction of the β-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of
czcCBA
mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.
Distinct Affinity of Binding Sites for S-Layer Homologous Domains in
Clostridium thermocellum
and
Bacillus anthracis
Cell Envelopes
Authors: Chauvaux Sylvie , Matuschek Markus , Beguin Pierre
Published: December 2019

Binding parameters were determined for the SLH (S-layer homologous) domains from the
Clostridium thermocellum
outer layer protein OlpB, from the
C. thermocellum
S-layer protein SlpA, and from the
Bacillus anthracis
S-layer proteins EA1 and Sap, using cell walls from
C. thermocellum
and
B. anthracis
. Each SLH domain bound to
C. thermocellum
and
B. anthracis
cell walls with a different

K
D

, ranging between 7.1 × 10
−7
and 1.8 × 10
−8
M. Cell wall binding sites for SLH domains displayed different binding specificities in
C. thermocellum
and
B. anthracis
. SLH-binding sites were not detected in cell walls of
Bacillus subtilis
. Cell walls of
C. thermocellum
lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from
C. thermocellum
cells by sodium dodecyl sulfate treatment, bound the SLH domains from
C. thermocellum
but not those from
B. anthracis
proteins. A corresponding component was not found in
B. anthracis
.
The Adsorption Protein Genes of
Xanthomonas campestris
Filamentous Phages Determining Host Specificity
Authors: Lin Nien-Tsung , Liu Tzu-Jun , Lee Tze-Ching , You Bih-Yuh , Yang Ming-Haw , Wen Fu-Shyan , Tseng Yi-Hsiung
Published: December 2019

Gene III (
gIII
) of φLf, a filamentous phage specifically infecting
Xanthomonas campestris
pv. campestris, was previously shown to encode a virion-associated protein (pIII) required for phage adsorption. In this study, the transcription start site for the gene and the N-terminal sequence of the protein were determined, resulting in the revision of the translation initiation site from the one previously predicted for this gene. For comparative study, the
gIII
of φXv, a filamentous phage specifically infecting
X. campestris
pv. vesicatoria, was cloned and sequenced. The deduced amino acid sequences of these two pIIIs exhibit a high degree of identity in their C-terminal halves and possess the structural features typical of the adsorption proteins of filamentous phages: a signal sequence in the N terminus, a long glycine-rich region near the center, and a hydrophobic membrane anchorage domain in the C terminus. The regions between
gIII
and the upstream
gVIII
, 128 nucleotides in both phages, are larger than those of other filamentous phages. A hybrid phage of φXv, consisting of the φLf pIII and all the other components derived from φXv, was able to infect
X. campestris
pv. campestris but not
X. campestris
pv. vesicatoria, indicating that
gIII
is the gene specifying host specificity and demonstrating the interchangeability of the pIIIs.
Analysis of an Autoregulatory Loop Controlling ToxT, Cholera Toxin, and Toxin-Coregulated Pilus Production in
Vibrio cholerae
Authors: Yu Rosa R. , DiRita Victor J.
Published: December 2019

Coordinate expression of many virulence genes in the human pathogen
Vibrio cholerae
is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activate
toxT
and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of
toxT
transcription. We tested a model whereby activation of
toxT
by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the
tcpA
promoter reads through a proposed terminator between the
tcpF
and
toxT
genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding
toxT
, one of which is longer than the other by 105 bp. Deletion of the
toxT
promoter (

toxT
Δpro

) resulted in the abolishment of
toxT
transcription, as predicted. Deletion of the
tcpA
promoter (

tcpA
Δpro

) had no effect on subsequent detection of the smaller
toxT
primer extension product, but the larger
toxT
product was not detected, indicating that this product may be the result of transcription from the
tcpA
promoter and not of initiation directly upstream of
toxT
. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. The

toxT
Δpro

strain produced little detectable CTX, while the

tcpA
Δpro

strain produced CTX levels intermediate between those of the wild-type and

toxT
Δpro

strains. Dependence of
toxT
transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The
tcpP
defect resulted in undetectable
toxT
transcription, whereas the
tcpH
mutation led to a diminishing of
toxT
RNA but not complete abolishment. Taken together, these results suggest that
toxT
transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.
The Lactate-Proton Symport of
Saccharomyces cerevisiae
Is Encoded by
JEN1
Authors: Casal Margarida , Paiva Sandra , Andrade Raquel P. , Gancedo Carlos , Leão Cecília
Published: December 2019

A mutant of
Saccharomyces cerevisiae
deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene
JEN1
that restored lactate transport. Disruption of
JEN1
abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in
S. cerevisiae
.
Assembly Requirements and Role of CotH during Spore Coat Formation in
Bacillus subtilis
Authors: Zilhão R. , Naclerio G. , Henriques A. O. , Baccigalupi L. , Moran C. P. , Ricca E.
Published: December 2019

We report Western blot data showing that the 42.8-kDa product of the previously characterized
cotH
locus (8) is a structural component of the
Bacillus subtilis
spore coat. We show that the assembly of CotH requires both CotE and GerE. In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.
The Type 2 Capsule Locus of
Streptococcus pneumoniae
Authors: Iannelli Francesco , Pearce Barbara J. , Pozzi Gianni
Published: December 2019

The type 2 capsule locus of
Streptococcus pneumoniae
was characterized in Avery’s strain D39, which is the parent strain of the standard transformation recipients currently used in pneumococcal research and is largely used as a virulent strain in studies on the pathogenesis of pneumococcal infections. The capsule locus was sequenced by using a 21.7-kb PCR fragment from the D39 genome as a template. Sequence data analysis showed the presence of 18 open reading frames, 17 of which have the same direction of transcription and all of which are potentially involved in capsule biosynthesis. It was also shown that R36A and R6, which are unencapsulated (rough) derivatives of D39, carry a 7,504-bp deletion involving nine capsule genes.
Benzene-Induced Uncoupling of Naphthalene Dioxygenase Activity and Enzyme Inactivation by Production of Hydrogen Peroxide
Authors: Lee Kyoung
Published: December 2019

Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-
cis
-(1
R
,2
S
)-1,2-dihydroxy-1,2-dihydronaphthalene with consumption of O
2
and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O
2
utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O
2
was detected as hydrogen peroxide. The rate of benzene-dependent O
2
consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O
2
consumption by NDO. Detailed experiments showed that the total amount of O
2
consumed and the rate of benzene-induced O
2
consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISP
NAP
) of NDO. Kinetic studies showed that ISP
NAP
was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISP
NAP
gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISP
NAP
containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O
2
consumption and as an inactivator of ISP
NAP
. It is proposed that the irreversible inactivation of ISP
NAP
occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (
·
OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [
14
C]benzene was used as the substrate,
cis
-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O
2
consumption and NADH oxidation.
The C-Terminal Fragment of the Precursor Tail Lysozyme of Bacteriophage T4 Stays as a Structural Component of the Baseplate after Cleavage
Authors: Kanamaru Shuji , Gassner Nadine C. , Ye Nanzhang , Takeda Shigeki , Arisaka Fumio
Published: December 2019

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in β-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.
Role of XDHC in Molybdenum Cofactor Insertion into Xanthine Dehydrogenase of
Rhodobacter capsulatus
Authors: Leimkühler Silke , Klipp Werner
Published: December 2019

Rhodobacter capsulatus
xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream of
xdhB
, a third gene was identified, designated
xdhC
, which is cotranscribed with
xdhAB
. Interposon mutagenesis revealed that the
xdhC
gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an α
2
β
2
heterotetramer in
R. capsulatus
. It was shown that XDHC neither is a transcriptional regulator for
xdh
gene expression nor influences XDH stability. To analyze the function of XDHC for XDH in
R. capsulatus
, inactive XDH was purified from an
xdhC
mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from the
xdhC
mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an
xdhC
mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion.
Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in
Rhodococcus rhodochrous
B276
Authors: Clark Daniel D. , Ensign Scott A.
Published: December 2019

The metabolism of acetone was investigated in the actinomycete
Rhodococcus rhodochrous
(formerly
Nocardia corallina
) B276. Suspensions of acetone- and isopropanol-grown
R. rhodochrous
readily metabolized acetone. In contrast,
R. rhodochrous
cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by
R. rhodochrous
was CO
2
dependent, and
14
CO
2
fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown
R. rhodochrous
by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.
Purification and Characterization of
Thermotoga maritima
Endonuclease IV, a Thermostable Apurinic/Apyrimidinic Endonuclease and 3′-Repair Diesterase
Authors: Haas Brian J. , Sandigursky Margarita , Tainer John A. , Franklin William A. , Cunningham Richard P.
Published: December 2019

An endonuclease IV homolog was identified as the product of a conceptual open reading frame in the genome of the hyperthermophilic bacterium
Thermotoga maritima
. The
T. maritima
endonuclease IV gene encodes a 287-amino-acid protein with 32% sequence identity to
Escherichia coli
endonuclease IV. The gene was cloned, and the expressed protein was purified and shown to have enzymatic activities that are characteristic of the endonuclease IV family of DNA repair enzymes, including apurinic/apyrimidinic endonuclease activity and repair activities on 3′-phosphates, 3′-phosphoglycolates, and 3′-
trans
-4-hydroxy-2-pentenal-5-phosphates. The
T. maritima
enzyme exhibits enzyme activity at both low and high temperatures. Circular dichroism spectroscopy indicates that
T. maritima
endonuclease IV has secondary structure similar to that of
E. coli
endonuclease IV and that the
T. maritima
endonuclease IV structure is more stable than
E. coli
endonuclease IV by almost 20°C, beginning to rapidly denature only at temperatures approaching 90°C. The presence of this enzyme, which is part of the DNA base excision repair pathway, suggests that thermophiles use a mechanism similar to that used by mesophiles to deal with the large number of abasic sites that arise in their chromosomes due to the increased rates of DNA damage at elevated temperatures.
YscP of
Yersinia pestis
Is a Secreted Component of the Yop Secretion System
Authors: Payne Patricia L. , Straley Susan C.
Published: December 2019

The
Yersinia pestis
low-Ca
2+
response stimulon is responsible for the environmentally regulated expression and secretion of antihost proteins (V antigen and Yops). We have previously shown that
yscO
encodes a secreted core component of the Yop secretion (Ysc) mechanism. In this study, we constructed and characterized in-frame deletions in the adjacent gene,
yscP
, in the
yscN–yscU
operon. The ΔP1 mutation, which removed amino acids 246 to 333 of YscP, had no effect on Yop expression or secretion, and the mutant protein, YscP1, was secreted, as was YscP in the parent. In contrast, the ΔP2 strain expressed and secreted less of each Yop than did the parent under the inductive conditions of 37°C and the absence of Ca
2+
, with an exception being YopE, which was only minimally affected by the mutation. The YscP2 protein, missing amino acids 57 to 324 of YscP, was expressed but not secreted by the ΔP2 mutant. The effect of the ΔP2 mutation was at the level of Yop secretion because YopM and V antigen still showed limited secretion when overproduced in
trans
. Excess YscP also affected secretion: overexpression of YscP in the parent, in either
yscP
mutant, or in an
lcrG
mutant effectively shut off secretion. However, co-overexpression of YscO and YscP had no effect on secretion, and YscP overexpression in an
lcrE
mutant had little effect on Yop secretion, suggesting that YscP acts, in conjunction with YscO, at the level of secretion control of LcrE at the bacterial surface. These findings place YscP among the growing family of mobile Ysc components that both affect secretion and themselves are secreted by the Ysc.
Expression of Two Glutathione
S
-Transferase Genes in the Yeast
Issatchenkia orientalis
Is Induced by
o
-Dinitrobenzene during Cell Growth Arrest
Authors: Tamaki Hisanori , Yamamoto Kenji , Kumagai Hidehiko
Published: December 2019

Glutathione
S
-transferases (GSTs) Y-1 and Y-2 from the yeast
Issatchenkia orientalis
were purified by passage through a glutathione-agarose column, and the cDNA for GST Y-1 was cloned and sequenced. The deduced amino acid sequence consisted of 188 residues with a total calculated molecular mass of 21,001 Da and showed 36.7% identity to that of GST Y-2, another GST isoenzyme expressed in this strain.
Escherichia coli
DH5α transformed with pUC119 harboring the GST Y-1 gene under the control of the
lac
promoter exhibited 29-fold-higher GST activity than the same strain with pUC119. Northern blot analysis revealed that both genes were highly expressed in cells cultured in the presence of 200 μM
o
-dinitrobenzene (DNB), one of the substrates of GST, while only the GST Y-1 gene was expressed, and only slightly, under normal (DNB-free) culture conditions. The DNB in the medium arrested cell growth until it was reduced by conjugation with reduced glutathione. Kinetic analysis of GST gene expression during detoxification of DNB revealed that the levels of expression of both genes were elevated within 3 h after the addition of DNB and that they further increased until 12 h postaddition. The levels of expression of both genes were decreased markedly when the DNB concentration in the culture medium was lowered. These results suggest that
I. orientalis
cells sense xenobiotics and arrest cell growth as a mechanism for preventing the induction of mutations by these compounds, while the levels of expression of the GST genes are up-regulated for detoxification.
The MobA-Linked Primase Is the Only Replication Protein of R1162 Required for Conjugal Mobilization
Authors: Henderson Dorian , Meyer Richard
Published: December 2019

Cells newly transformed with plasmid R1162 DNA were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer. An intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells.
First Isolation and Cultivation of
Borrelia burgdorferi
Sensu Lato from Missouri
Authors: Oliver J. H. , Kollars T. M. , Chandler F. W. , James A. M. , Masters E. J. , Lane R. S. , Huey L. O.
Published: December 2019

Five
Borrelia burgdorferi
sensu lato isolates from Missouri are described. This represents the first report and characterization of such isolates from that state. The isolates were obtained from either
Ixodes dentatus
or
Amblyomma americanum
ticks that had been feeding on cottontail rabbits (
Sylvilagus floridanus
) from a farm in Bollinger County, Mo., where a human case of Lyme disease had been reported. All isolates were screened immunologically by indirect immunofluorescence by using monoclonal antibodies to
B. burgdorferi
-specific outer surface protein A (OspA) (antibodies H3TS and H5332),
B. burgdorferi
-specific OspB (antibody H6831),
Borrelia
(genus)-specific antiflagellin (antibody H9724), and
Borrelia hermsii
-specific antibody (antibody H9826). Analysis of the isolates also involved a comparison of their protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the isolates were analyzed by PCR with six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain
B. burgdorferi
B-31. Although some genetic variability was detected among the five isolates as well as between them and the B-31 strain, enough similarities were found to classify them as
B. burgdorferi
sensu lato.
Past and Present Hepatitis G Virus Infections in Areas Where Hepatitis C is Highly Endemic and Those Where It Is Not Endemic
Authors: Tanaka Eiji , Tacke Michael , Kobayashi Masakazu , Nakatsuji Yoshiyuki , Kiyosawa Kendo , Schmolke Susanne , Engel Alfred M. , Hess Georg , Alter Harvey J.
Published: December 2019

We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5′ noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (
P
< 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (
P
< 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (
P
< 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.
Enterobacterial Repetitive Intergenic Consensus Sequences as Molecular Targets for Typing of
Mycobacterium tuberculosis
Strains
Authors: Sechi Leonardo A. , Zanetti Stefania , Dupré Ilaria , Delogu Giovanni , Fadda Giovanni
Published: December 2019

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of
Mycobacterium tuberculosis
; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of
M. tuberculosis
was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS
6110
and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS
6110
fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of
M. tuberculosis
infections, especially when IS
6110
fingerprinting is not of any help.
Detection of
Porphyromonas gingivalis
from Saliva by PCR by Using a Simple Sample-Processing Method
Authors: Mättö Jaana , Saarela Maria , Alaluusua Satu , Oja Virva , Jousimies-Somer Hannele , Asikainen Sirkka
Published: December 2019

Simple sample-processing methods for PCR detection of
Porphyromonas gingivalis
, a major pathogen causing adult periodontitis, from saliva were studied. The ability to detect
P. gingivalis
from 118 salivary samples by PCR after boiling and Chelex 100 processing was compared with bacterial culture.
P. gingivalis
was detected three times more often by PCR than by culture. Chelex 100 processing of saliva proved to be effective in preventing PCR inhibition and was applied to determine the occurrence of
P. gingivalis
in saliva samples from 263 Finnish subjects between 5 and 80 years of age. The occurrence of
P. gingivalis
increased with age, and it was detected by PCR in the saliva of 5.0% of subjects between 5 and 10 years of age, 13.8% of subjects between 11 and 20 years of age, 13.4% of subjects between 21 and 30 years of age, and 63.3% of subjects between 31 and 80 years of age. The results indicate that
P. gingivalis
is a rare finding in saliva from periodontally healthy children and young adults but a frequent one in saliva from adult periodontitis patients.
An Internal Control for Routine Diagnostic PCR: Design, Properties, and Effect on Clinical Performance
Authors: Rosenstraus Maurice , Wang Zhuang , Chang Sheng-Yung , DeBonville David , Spadoro Joanne P.
Published: December 2019

We constructed internal controls (ICs) to provide assurance that clinical specimens are successfully amplified and detected. The IC nucleic acids contain primer binding regions identical to those of the target sequence and contain a unique probe binding region that differentiates the IC from amplified target nucleic acid. Because only 20 copies of the IC are introduced into each test sample, a positive IC signal indicates that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity. The COBAS AMPLICOR
Chlamydia trachomatis
,
Neisseria gonorrhoeae
,
Mycobacterium tuberculosis
, and human hepatitis C virus tests exhibited inhibition rates ranging from 5 to 9%. Approximately 64% of these inhibitory specimens were not inhibitory when a second aliquot was tested. Because repeatedly inhibitory specimens were not reported as false negative and because additional infected specimens were detected during retesting, test sensitivities were 1 to 6% greater than they would have been if the IC had not been used.
In Vitro Activity of the New Triazole Voriconazole (UK-109,496) against Opportunistic Filamentous and Dimorphic Fungi and Common and Emerging Yeast Pathogens
Authors: Espinel-Ingroff Ana
Published: December 2019

The in vitro antifungal activity of a new triazole derivative, voriconazole, was compared with those of itraconazole and amphotericin B against 67 isolates ofAspergillus flavus,Aspergillus fumigatus,Bipolarisspp.,Fusarium oxysporum,Fusarium solani,Pseudallescheria boydii,Rhizopus arrhizus,Blastomyces dermatitidis,Histoplasma capsulatum, andSporothrix schenckii. The in vitro activities of voriconazole were also compared with those of amphotericin B, fluconazole, and itraconazole against 189 isolates of emerging and common yeast pathogens ofBlastoschizomyces capitatus,Candida(13 species),Cryptococcus neoformans,Hansenula anomala,Rhodotorula rubra,Saccharomyces cerevisiae,Sporobolomyces salmonicolor, andTrichosporon beigelii. MICs were determined according to a procedure under evaluation by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution testing of filamentous fungi and by the NCCLS M27-A broth microdilution method for yeasts. The in vitro activities of voriconazole were similar to or better than those of itraconazole and amphotericin B againstAspergillusspp.,Fusariumspp., andP. boydiias well as againstB. dermatitidisandH. capsulatum. The activities of voriconazole were also comparable to or better than those of amphotericin B, fluconazole, and itraconazole against most species of yeasts tested. Exceptions were certain isolates ofR. rubraandS. salmonicolor. These results suggest that voriconazole has a wide spectrum of activity in vitro; its effectiveness in the treatment of human mycoses is under evaluation in clinical trials.
Characterization of
rpoB
Mutations in Rifampin-Resistant Clinical
Mycobacterium tuberculosis
Isolates from Greece
Authors: Matsiota-Bernard Peggy , Vrioni Georgia , Marinis Evangelos
Published: December 2019

There is a geographic distribution of
Mycobacterium tuberculosis
strains with various
rpoB
gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify
rpoB
mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect
rpoB
mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct
rpoB
mutations in codons Ser
531
, His
526
, and Asp
516
were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser
531
and His
526
accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related
M. tuberculosis
strains can have subclones bearing distinct mutant
rpoB
alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in
M. tuberculosis
before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.
Antitoxin-in-Membrane and Antitoxin-in-Well Assays for Detection of Toxigenic
Corynebacterium diphtheriae
Authors: Reinhardt D. J. , Lee A. , Popovic T.
Published: December 2019

The Elek culture plate precipitin test is routinely used for the detection of exotoxin from toxigenic strains of
Corynebacterium diphtheriae
. Recently, the World Health Organization standardized this test to ensure accuracy, reliability, and reproducibility. In this study, we further modified the standard Elek test by using the antitoxin-in-membrane (AIM) and antitoxin-in-well (AIW) approaches. In the AIM tests, each strain was stabbed and streaked backwards and away from a point approximately 7 mm from the edge of a sterile cellulose acetate-cellulose nitrate filter membrane disk (pore size, 0.45 μm; diameter, 25 mm) containing 25 IU of diphtheria antitoxin. For AIW tests, a central well (diameter, 5 mm) containing 9 μl of antitoxin (4.5 IU) was surrounded by eight equidistant stab-streaks of each strain placed 10 mm from the well. In both methods, precipitin bands of identity typically were noted after 24- and 48-h incubations at 37°C. Both toxigenic and weak toxigenic strains gave clear and reproducible results. Compared with the standard Elek test, the AIM and AIW tests each use 50% less medium and 75 and 87% less antitoxin, respectively. AIM has the potential to test up to 14 isolates and AIW has the potential to test up to 24 isolates on the same plate. Furthermore, clearer positives were noted with weak toxigenic strains. In a blinded test of 209 verified
C. diphtheriae
isolates, a 99.5% agreement with the standard Elek test was obtained overall. Both modifications conserve reagents and medium, permit the simultaneous testing of a larger number of strains, and may be particularly suitable for reference laboratories or hospitals involved in diphtheria epidemic settings.
Repetitive Sequence-Based PCR versus Pulsed-Field Gel Electrophoresis for Typing of
Enterococcus faecalis
at the Subspecies Level
Authors: Malathum Kumthorn , Singh Kavindra V. , Weinstock George M. , Murray Barbara E.
Published: December 2019

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate
Enterococcus faecalis
isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in
Streptococcus pneumoniae
, was applied to 41 isolates of
E. faecalis
collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were β-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.
Hepatitis C Virus Heteroduplex Tracking Assay for Genotype Determination Reveals Diverging Genotype 2 Isolates in Italian Hemodialysis Patients
Authors: Calvo Pier Luigi , Kansopon Joe , Sra Kuldip , Quan Stella , DiNello Robert , Guaschino Roberto , Calabrese Giovanni , Danielle Franca , Brunetto Mauizia Rossana , Bonino Ferruccio , Massaro Anna Lucia , Polito Alan , Houghton Michael , Weiner Amy J.
Published: December 2019

A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT)-PCR products from 15 Italian dialysis patients confirmed the specificity and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) 1b, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections. The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/or restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype 1a, 1b, 2a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples that were designated non-2a or 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5′ untranslated region. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the population being studied. Serotyping (RIBA serotyping strip immunoblot assay kit) of 23 dialysis patients showed that the genotype could be determined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR-positive samples, indicating that the two tests complement each other.
Comparison of Six Sets of PCR Primers from Two Different Genomic Regions for Amplification of GB Virus C/Hepatitis G Virus RNA
Authors: Andonov Anton , Sauder Connie , Jacobsen Heather , Chaudhary Rabindra
Published: December 2019

Forty-four clinical samples positive for GB virus C (GBV-C)/hepatitis G virus (HGV) were tested with six primer sets, four from the 5′ untranslated region (5′-UTR) and two from the NS5a genomic region. Two of the 5′-UTR primer sets, when used in a single-round 60-cycle PCR, detected between 86.4 and 97.7% of the positive samples, while two different sets from the same area, when used in a nested PCR, amplified between 97.7 and 100% of the positive specimens. Both sets from the NS5a region, when used in a single-round PCR, detected 95.5% of the GBV-C/HGV-positive samples. Parallel testing with two PCR sets, one from the 5′-UTR and a second from NS5a, may eliminate false-negative results attributable to the genetic heterogeneity of the virus.