bluuebunny/crossref_metadata_2025 huggingface dataset:
Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast
Saccharomyces cerevisiae
Authors: Athenstaedt Karin , Weys Sabine , Paltauf Fritz , Daum Günther
Published: December 2019

In the yeast
Saccharomyces cerevisiae
lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the
gat1
mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-
3
H, U-
14
C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the
gat1
mutant and the 1-acylglycerol-3-phosphate acyltransferase
slc1
mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.
Molecular Cloning, Sequencing, and Expression of a Novel Multidomain Mannanase Gene from
Thermoanaerobacterium polysaccharolyticum
Authors: Cann Isaac K. O. , Kocherginskaya Svetlana , King Michael R. , White Bryan A. , Mackie Roderick I.
Published: December 2019

The
manA
gene of
Thermoanaerobacterium polysaccharolyticum
was cloned in
Escherichia coli
. The open reading frame of
manA
is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative −35 (TTCGC) and −10 (TAAAAT) promoter sequence. The ManA of
T. polysaccharolyticum
is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the
Bacillus-Clostridium
cluster. The ManA of
T. polysaccharolyticum
, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.
Rapid Hypothesis Testing with
Candida albicans
through Gene Disruption with Short Homology Regions
Authors: Wilson R. Bryce , Davis Dana , Mitchell Aaron P.
Published: December 2019

Disruption of newly identified genes in the pathogen
Candida albicans
is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of
C. albicans
genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes,
ARG5
and
ADE2
, and two sequences newly identified through the
Candida
genome project,
HRM101
and
ENX3. HRM101
and
ENX3
are homologous to genes in the conserved
RIM101
(previously called
RIM1
) and
PacC
pathways of
Saccharomyces cerevisiae
and
Aspergillus nidulans
. We show that three independent
hrm101/hrm101
mutants and two independent
enx3/enx3
mutants are defective in filamentation on Spider medium. These observations argue that
HRM101
and
ENX3
sequences are indeed portions of genes and that the respective gene products have related functions.
Catabolic Repression of
secB
Expression Is Positively Controlled by Cyclic AMP (cAMP) Receptor Protein-cAMP Complexes at the Transcriptional Level
Authors: Seoh Hyuk Kyu , Tai Phang C.
Published: December 2019

SecB, a protein export-specific chaperone, enhances the export of a subset of proteins across cytoplasmic membranes of
Escherichia coli
. Previous studies showed that the synthesis of SecB is repressed by the presence of glucose in the medium. The derepression of SecB requires the products of both the
cya
and
crp
genes, indicating that
secB
expression is under the control of catabolic repression. In this study, two
secB
-specific promoters were identified. In addition, 5′ transcription initiation sites from these two promoters were determined by means of
secB-lacZ
fusions and primer extension. The distal P1 promoter appeared to be independent of carbon sources, whereas the proximal P2 promoter was shown to be subject to control by the cyclic AMP (cAMP) receptor protein (CRP)-cAMP complexes. Gel-mobility shift studies showed that this regulation results from direct interaction between the
secB
P2 promoter region and the CRP-cAMP complex. Moreover, the CRP binding site on the
secB
gene was determined by DNase I footprinting and further substantiated by mutational analysis. The identified
secB
CRP binding region is centered at the −61.5 region of the
secB
gene and differed from the putative binding sites predicted by computer analysis.
SodA and Manganese Are Essential for Resistance to Oxidative Stress in Growing and Sporulating Cells of
Bacillus subtilis
Authors: Inaoka Takashi , Matsumura Yoshinobu , Tsuchido Tetsuaki
Published: December 2019

We constructed a
sodA
-disrupted mutant of
Bacillus subtilis
168, BK1, by homologous recombination. The mutant was not able to grow in minimal medium without Mn(II). The spore-forming ability of strain BK1 was significantly lower in Mn(II)-depleted medium than that of the wild-type strain. These deleterious effects caused by the
sodA
mutation were reversed when an excess of Mn(II) was used to supplement the medium. Moreover, the growth inhibition by superoxide generators in strain BK1 and its parent strain was also reversed by the supplementation with excess Mn(II). We therefore estimated the Mn-dependent superoxide-scavenging activity in BK1 cells. Whereas BK1 cells have no detectable superoxide dismutase (Sod) on native gel, the superoxide-scavenging activity in crude extracts of BK1 cells grown in Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) was significantly detected by the modified Sod assay method without using EDTA. The results obtained suggest that Mn, as a free ion or a complex with some cellular component, can catalyze the elimination of superoxide and that both SodA and Mn(II) are involved not only in the superoxide resistance of vegetative cells but also in sporulation.
Functional Analysis of the Active Partition Region of the
Coxiella burnetii
Plasmid QpH1
Authors: Lin Zhicheng , Mallavia Louis P.
Published: December 2019

The partition region
qsopAB
of the
Coxiella burnetii
plasmid QpH1 was analyzed. Locus
qsopA
alone appears to fulfill the partitioning function; QsopA represses its own promoter 17-fold. Two partition-associated incompatibility sites were identified:
incA
in a 200-bp region covering the
qsopA
promoter and
incB
in the
qsopB
locus.
Characterization of the Pyoluteorin Biosynthetic Gene Cluster of
Pseudomonas fluorescens
Pf-5
Authors: Nowak-Thompson Brian , Chaney Nancy , Wing Jenny S. , Gould Steven J. , Loper Joyce E.
Published: December 2019

Ten genes (
plt
) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of
Pseudomonas fluorescens
Pf-5. The deduced amino acid sequences of eight
plt
genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (
pltB
,
pltC
), an acyl coenzyme A (acyl-CoA) dehydrogenase (
pltE
), an acyl-CoA synthetase (
pltF
), a thioesterase (
pltG
), and three halogenases (
pltA
,
pltD
, and
pltM
). Insertions of the transposon Tn
5
or Tn
3
-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight
plt
products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another
plt
gene,
pltR
, is similar to those of members of the LysR family of transcriptional activators.
pltR
and
pltM
are transcribed divergently from the
pltLABCDEFG
gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating
pltRM
from
pltLABCDEFG
. Transcription of the pyoluteorin biosynthesis genes
pltB
,
pltE
, and
pltF
, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a
pltR
mutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.
RsaL, a Novel Repressor of Virulence Gene Expression in
Pseudomonas aeruginosa
Authors: de Kievit Teresa , Seed Patrick C. , Nezezon Jonathon , Passador Luciano , Iglewski Barbara H.
Published: December 2019

As components of a
Pseudomonas aeruginosa
quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second
P. aeruginosa
quorum-sensing system. To date, no information exists on negative regulation of the quorum-sensing cascade in
P. aeruginosa.
Here we describe a novel gene,
rsaL
, which is located downstream from
lasR
and transcribed antisense relative to
lasR.
In
P. aeruginosa,
overexpression of
rsaL
results in reduced
lasB
expression and decreased elastase activity. With the use of a six-His protein fusion system, we demonstrate that
rsaL
encodes an 11-kDa protein. Direct quantitation of PAI-1 levels in cultures and studies utilizing
Escherichia coli
lambda lysogens carrying
lacZ
transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene,
lasI
. RsaL’s repressive effect on
lasI
and the associated decrease in elastase activity have important implications for the expression of all LasR–PAI-1-dependent virulence genes and the overall pathogenicity of
P. aeruginosa.
Target Joining of Duplicated Insertion Sequence IS
21
Is Assisted by IstB Protein In Vitro
Authors: Schmid Sergio , Berger Bernard , Haas Dieter
Published: December 2019

Tandemly repeated insertion sequence IS
21
, located on a suicide plasmid, promoted replicon fusion with bacteriophage λ in vitro in the presence of ATP. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS
21
after both proteins had been overproduced in
Escherichia coli
. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS
21
.
RecD Function Is Required for High-Pressure Growth of a Deep-Sea Bacterium
Authors: Bidle Kelly A. , Bartlett Douglas H.
Published: December 2019

A genomic library derived from the deep-sea bacterium
Photobacterium profundum
SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533–7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to
recD
, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in
Escherichia coli recD
mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the
recD
gene. Two additional
recD
mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3′ truncation of the
recD
coding sequence. Surprisingly, the introduction of the SS9
recD
gene into an
E. coli recD
mutant had two dramatic effects. At high pressure, SS9
recD
enabled growth in the
E. coli
mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type
E. coli recD
. These results suggest that the SS9
recD
gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.
mrp
, a Multigene, Multifunctional Locus in
Bacillus subtilis
with Roles in Resistance to Cholate and to Na
+
and in pH Homeostasis
Authors: Ito Masahiro , Guffanti Arthur A. , Oudega Bauke , Krulwich Terry A.
Published: December 2019

A 5.9-kb region of the
Bacillus subtilis
chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins. Homologues of the first gene of this operon, for which the designation
mrp
(multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na
+
/H
+
antiporter or a K
+
/H
+
antiporter. In the present studies of the
B. subtilis mrp
operon, both polar and nonpolar mutations in
mrpA
were generated. Growth of these mutants was completely inhibited by concentrations of added Na
+
as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K
+
. A null mutant that was constructed by full replacement of the
mrp
operon was even more Na
+
sensitive. A double mutant with mutations in both
mrpA
and the multifunctional antiporter-encoding
tetA
(L) gene was no more sensitive than the
mrpA
mutants to Na
+
, consistent with a major role for
mrpA
in Na
+
resistance. Expression of
mrpA
from an inducible promoter, upon insertion into the
amyE
locus, restored significant Na
+
resistance in both the polar and nonpolar
mrpA
mutants but did not restore resistance in the null mutant. The
mrpA
disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na
+
and, to some extent, K
+
in the range from 10 to 25 mM. By contrast, the
mrpA tetA
(L) double mutant, like the
tetA
(L) single mutant, completely lost its capacity for both Na
+
- and K
+
-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus,
tetA
(L) has a more pronounced involvement than
mrpA
in pH regulation. Measurements of Na
+
efflux from the wild-type strain, the nonpolar
mrpA
mutant, and the complemented mutant indicated that inducible expression of
mrpA
increased the rate of protonophore- and cyanide-sensitive Na
+
efflux over that in the wild-type in cells preloaded with 5 mM Na
+
. The
mrpA
and null mutants showed no such efflux in that concentration range. This is consistent with MrpA encoding a secondary, proton motive force-energized Na
+
/H
+
antiporter. Studies of a polar mutant that leads to loss of
mrpFG
and its complementation in
trans
by
mrpF
or
mrpFG
support a role for MrpF as an efflux system for Na
+
and cholate. Part of the Na
+
efflux capacity of the whole
mrp
operon products is attributable to
mrpF
. Neither
mrpF
nor
mrpFG
expression in
trans
enhanced the cholate or Na
+
resistance of the null mutant. Thus, one or more other
mrp
gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in
trans
. Also, phenotypic differences among the
mrp
mutants suggest that functions in addition to Na
+
and cholate resistance and pH homeostasis will be found among the remaining
mrp
genes.
kdgR
Ecc

Negatively Regulates Genes for Pectinases, Cellulase, Protease, Harpin
Ecc
, and a Global RNA Regulator in
Erwinia carotovora
subsp.
carotovora
Authors: Liu Yang , Jiang Guoqiao , Cui Yaya , Mukherjee Asita , Ma Wei Lei , Chatterjee Arun K.
Published: December 2019

Erwinia carotovora
subsp.
carotovora
produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium.
E. carotovora
subsp.
carotovora
also produces Harpin
Ecc
, the elicitor of the hypersensitive reaction. We document here that KdgR
Ecc
(Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the
E. chrysanthemi
repressor, KdgR
Ech
and the
Escherichia coli
repressor, KdgR
Eco
, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and Harpin
Ecc
production in
E. carotovora
subsp.
carotovora
. The levels of
pel-1
,
peh-1
,
celV
, and

hrpN
Ecc

transcripts are markedly affected by KdgR
Ecc
. The KdgR
Ecc
−
mutant is more virulent than the KdgR
Ecc
+
parent. Thus, our data for the first time establish a global regulatory role for KdgR
Ecc
in
E. carotovora
subsp.
carotovora
. Another novel observation is the negative effect of KdgR
Ecc
on the transcription of
rsmB
(previously
aepH
), which specifies an RNA regulator controlling exoenzyme and Harpin
Ecc
production. The levels of
rsmB
RNA are higher in the KdgR
Ecc
−
mutant than in the KdgR
Ecc
+
parent. Moreover, by DNase I protection assays we determined that purified KdgR
Ecc
protected three 25-bp regions within the transcriptional unit of
rsmB
. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgR
Ecc
-binding site as 5′-G/AA/TA/TG
AAA
[N
6
]
TTT
CAG/TG/TA-3′. Two such KdgR
Ecc
-binding sites occur in
rsmB
DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgR
Ecc
-binding site within nucleotides +207 and +231. Analysis of
lacZ
transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of
rsmB
. KdgR
Ecc
also binds the operator DNAs of
pel-1
and
peh-1
genes and represses expression of a
pel1-lacZ
and a
peh1-lacZ
transcriptional fusions. We conclude that KdgR
Ecc
affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgR
Ecc
affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.
The CtrA Response Regulator Mediates Temporal Control of Gene Expression during the
Caulobacter
Cell Cycle
Authors: Reisenauer Ann , Quon Kim , Shapiro Lucy
Published: December 2019

In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the
Caulobacter crescentus
cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the
fliQ
flagellar gene and the
ccrM
DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. P
fliQ
is activated earlier than P
ccrM
. Phosphorylated CtrA (CtrA∼P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for P
fliQ
. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in P
ccrM
significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of
ccrM
expression. Our data indicate that differences in the affinity of CtrA∼P for P
fliQ
and P
ccrM
regulate, in part, the temporal expression of these genes. However, the timing of
fliQ
transcription but not of
ccrM
transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA∼P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA∼P and its interaction with accessory proteins influence the temporal expression of
fliQ
,
ccrM
, and other key cell cycle genes and ultimately the regulation of the cell cycle.
Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal
bop
Gene Promoter
Authors: Baliga Nitin S. , DasSarma Shiladitya
Published: December 2019

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal
bop
promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized
bop
promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon
Halobacterium
sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum
−
bop
)
Halobacterium
mutant for purple (Pum
+
bop
+
) colonies on agar plates and analyzed for
bop
mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for
bop
promoter activity.
A Region Near the C-Terminal End of
Escherichia coli
DNA Helicase II Is Required for Single-Stranded DNA Binding
Authors: Mechanic Leah E. , Latta Marcy E. , Matson Steven W.
Published: December 2019

The role of the C terminus of
Escherichia coli
DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDΔ107C (deletion of the last 107 C-terminal amino acids), UvrDΔ102C, and UvrDΔ40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDΔ107C and UvrDΔ102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDΔ40C protein fully complemented the loss of helicase II in both repair pathways. UvrDΔ102C and UvrDΔ40C were purified to apparent homogeneity and characterized biochemically. UvrDΔ102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (
k
cat
= 0.01% of the wild-type level). UvrDΔ40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.
Genetic Analysis of the Mobilization and Leading Regions of the IncN plasmids pKM101 and pCU1
Authors: Paterson E. Suzanne , Moré Margret I. , Pillay Gansen , Cellini Christina , Woodgate Roger , Walker Graham C. , Iyer V. N. , Winans Stephen C.
Published: December 2019

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical
oriT
sequences as well as conserved restriction endonuclease cleavage patterns within their
tra
regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes,
traI
,
traJ
, and
traK
, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (
oriT
). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three
tra
complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both
traK
and
traI
were required for efficient interplasmid site-specific recombination at
oriT
, while
traJ
was not required. The leading region of pKM101 contains three genes (
stbA
,
stbB
, and
stbC
), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.
In Vitro Synthesis of Peptidoglycan Precursors Modified with
N
-Acetylputrescine by
Cyanophora paradoxa
Cyanelle Envelope Membranes
Authors: Pfanzagl Beatrix , Löffelhardt Wolfgang
Published: December 2019

The photosynthetic organelles (cyanelles) of the protist
Cyanophora paradoxa
are surrounded by a peptidoglycan wall, modified through amidation with
N
-acetylputrescine. Cyanelle envelope membrane preparations were shown to catalyze the lipid-linked steps of peptidoglycan biosynthesis as well as the putrescinylation and subsequent acetylation, occurring at the stage of lipid I and/or lipid II.
DnaA Boxes Are Important Elements in Setting the Initiation Mass of
Escherichia coli
Authors: Christensen Bjarke Bak , Atlung Tove , Hansen Flemming G.
Published: December 2019

The binding of DnaA protein to its DNA binding sites—DnaA boxes—in the chromosomal
oriC
region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the
oriC
region into
Escherichia coli
and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per
oriC
locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per
oriC
locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in
E. coli
cells.
Isoaspartate in Ribosomal Protein S11 ofEscherichia coli
Authors: David Cynthia L. , Keener John , Aswad Dana W.
Published: December 2019

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its β-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme proteinl-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal α-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid intoEscherichia colithat allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels inE. coliproteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case;E. colicells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins inE. colidiffered dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.
Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis
Authors: Cousins Debby , Williams Suzette , Liébana Ernesto , Aranaz Alicia , Bunschoten Annelies , Van Embden Jan , Ellis Trevor
Published: December 2019

DNA fingerprinting techniques were used to type 273 isolates of
Mycobacterium bovis
from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS
6110
, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of
Mycobacterium tuberculosis
(J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS
6110
, and 81.5% of these carried IS
6110
on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS
6110
identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS
6110
was sufficiently sensitive for the typing of isolates with more than three copies of IS
6110
, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS
6110
. Spoligotyping may have advantages for the rapid typing of
M. bovis
, but it needs to be made more sensitive.
Evaluation of the OptiMAL Test for Rapid Diagnosis of
Plasmodium vivax
and
Plasmodium falciparum
Malaria
Authors: Palmer Carol J. , Lindo John F. , Klaskala Winslow I. , Quesada Jose A. , Kaminsky Rina , Baum Marianna K. , Ager Arba L.
Published: December 2019

The development of rapid and specific diagnostic tests to identify individuals infected with malaria is of paramount importance in efforts to control the severe public health impact of this disease. This study evaluated the ability of a newly developed rapid malaria diagnostic test, OptiMAL (Flow Inc., Portland, Oreg.), to detect
Plasmodium vivax
and
Plasmodium falciparum
malaria during an outbreak in Honduras. OptiMAL is a rapid (10-min) malaria detection test which utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme parasite lactate dehydrogenase (pLDH). Differentiation of malaria parasites is based on antigenic differences between the pLDH isoforms. Since pLDH is produced only by live
Plasmodium
parasites, this test has the ability to differentiate live from dead organisms. Results from the OptiMAL test were compared to those obtained by reading 100 fields of traditional Giemsa-stained thick-smear blood films. Whole-blood samples were obtained from 202 patients suspected of having malaria. A total of 96 samples (48%) were positive by blood films, while 91 (45%) were positive by the OptiMAL test. The blood films indicated that 82% (79 of 96) of the patients were positive for
P. vivax
and 18% (17 of 96) were infected with
P. falciparum
. The OptiMAL test showed that 81% (74 of 91) were positive for
P. vivax
and 19% (17 of 91) were positive for
P. falciparum
. These results demonstrated that the OptiMAL test had sensitivities of 94 and 88% and specificities of 100 and 99%, respectively, when compared to traditional blood films for the detection of
P. vivax
and
P. falciparum
malaria. Blood samples not identified by OptiMAL as malaria positive normally contained parasites at concentrations of less than 100/μl of blood. Samples found to contain
P. falciparum
were further tested by two other commercially available rapid malaria diagnostic tests, ParaSight-F (Becton Dickinson, Cockeysville, Md.) and ICT Malaria P.f. (ICT Diagnostics, Sydney, Australia), both of which detect only
P. falciparum
. Only 11 of the 17 (65%)
P. falciparum
-positive blood samples were identified by the ICT and ParaSight-F tests. Thus, OptiMAL correctly identified
P. falciparum
malaria parasites in patient blood samples more often than did the other two commercially available diagnostic tests and showed an excellent correlation with traditional blood films in the identification of both
P. vivax
malaria and
P. falciparum
malaria. We conclude that the OptiMAL test is an effective tool for the rapid diagnosis of malaria.
Evidence That the Enterotoxin Gene Can Be Episomal inClostridium perfringensIsolates Associated with Non-Food-Borne Human Gastrointestinal Diseases
Authors: Collie Renee E. , McClane Bruce A.
Published: December 2019

Clostridium perfringensenterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of humanC. perfringenstype A food poisoning. CPE-producingC. perfringensisolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43cpe-positiveC. perfringensisolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomalcpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry theircpeon an episome. Collectively, these results provide the first evidence that distinct subpopulations ofcpe-positiveC. perfringensisolates may be responsible forC. perfringenstype A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys,cpeRFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations ofcpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.
Case of Triple Endocarditis Caused by
Rothia dentocariosa
and Results of a Survey in France
Authors: Kong Robert , Mebazaa Alexandre , Heitz Bertrand , De Briel Dominique André , Kiredjian Martine , Raskine Laurent , Payen Didier
Published: December 2019

This is the first case of endocarditis in which
Rothia dentocariosa
was cultured from three affected valves. In addition, the records of two microbiology laboratories in France showed that
R. dentocariosa
was rarely involved in severe infection and that positive blood cultures were not associated with endocarditis.
Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase
Authors: Poyart Claire , Quesne Gilles , Coulon Stephane , Berche Patrick , Trieu-Cuot Patrick
Published: December 2019

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (

sodA
int

) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (
S. acidominimus
,
S. agalactiae
,
S. alactolyticus
,
S. anginosus
,
S. bovis
,
S. constellatus
,
S. canis
,
S. cricetus
,
S. downei
,
S. dysgalactiae
,
S. equi
subsp.
equi
,
S. equi
subsp.
zooepidemicus
,
S. equinus
,
S. gordonii
,
S. iniae
,
S. intermedius
,
S. mitis
,
S. mutans
,
S. oralis
,
S. parasanguis
,
S. pneumoniae
,
S. porcinus
,
S. pyogenes
,
S. salivarius
,
S. sanguis
,
S. sobrinus
,
S. suis
,
S. thermophilus
, and
S. vestibularis
). Phylogenetic analysis of these

sodA
int

fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their

sodA
int

fragments on the phylogenetic tree of the

sodA
int

fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
Detection of Human Herpesvirus 6 DNA in Serum by a Microplate PCR-Hybridization Assay
Authors: Osiowy Carla , Prud’homme Isabelle , Monette Michael , Zou Shimian
Published: December 2019

PCR was performed on DNA extracts derived from clinical serum samples submitted for human herpesvirus 6 (HHV-6) serological examination. To detect amplified HHV-6 products, a hybridization-based microtiter plate assay (PCR ELISA; Boehringer Mannheim) was used. The assay system was found to be rapid, specific, and sensitive. Approximately three copies of a plasmid-based HHV-6 sequence could be detected, and no cross amplification was observed with HHV-7 genomic DNA. There was no correlation found between HHV-6 DNA detection and serological status in clinical serum samples from individuals more than 2 years old. On the other hand, in serum samples from infants less than 2 years old, a high rate of detection of HHV-6 DNA was observed in those who lacked immunoglobulin G and M antibodies to HHV-6 (55%). In this regard, PCR of serum DNA extracts may be used as a sensitive indicator of active HHV-6 infection in infants prior to their seroconversion.
Pre-AIDS Era Isolates ofPneumocystis cariniif. sp.hominis: High Genotypic Similarity with Contemporary Isolates
Authors: Tsolaki Anthony G. , Beckers Pieter , Wakefield Ann E.
Published: December 2019

Isolates ofPneumocystis cariniif. sp.hominiswere examined from six individuals who died ofP. cariniipneumonia between 1968 and 1981 and who had underlying immunodeficiencies which were not due to human immunodeficiency virus infection. DNA sequence variation was analyzed in the genes encoding the mitochondrial large subunit rRNA (mt LSU rRNA), the internal transcribed spacer (ITS) regions of the nuclear rRNA, thearomlocus, and the mitochondrial small subunit rRNA. No major variations were observed when these isolates were compared to isolates from HIV-infected individuals. A small number of minor differences were detected. A new position at which variation occurred in the mt LSU rRNA was observed in one sample. Three new ITS sequence types were identified. A total of nine different ITS sequence types were found in the six samples. Mixed infection with different ITS sequence types ofP. cariniif. sp.hominiswas observed in four of the six samples. The ITS locus was the most informative of the four loci for distinguishing among the isolates ofP. cariniif. sp.hominis. The data suggest that isolates ofP. cariniif. sp.hominisfrom before the AIDS pandemic are genetically very similar to those currently found in HIV-infected individuals.
Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate
Authors: Fredricks David N. , Relman David A.
Published: December 2019

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with
Escherichia coli
was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted
E. coli
16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.
Human Exposure to a Granulocytic Ehrlichia and Other Tick-Borne Agents in Connecticut
Authors: Magnarelli Louis A. , Ijdo Jacob W. , Anderson John F. , Padula Steven J. , Flavell Richard A. , Fikrig Erol
Published: December 2019

Indirect fluorescent-antibody (IFA) staining methods with
Ehrlichia equi
(MRK or BDS strains) and Western blot analyses containing a human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain) were used to confirm probable human cases of infection in Connecticut during 1995 and 1996. Also included were other tests for
Ehrlichia chaffeensis
, the agent of human monocytic ehrlichiosis (HME),
Babesia microti
, and
Borrelia burgdorferi
. Thirty-three (8.8%) of 375 patients who had fever accompanied by marked leukopenia or thrombocytopenia were serologically confirmed as having HGE. Western blot analyses of a subset of positive sera confirmed the results of the IFA staining methods for 15 (78.9%) of 19 seropositive specimens obtained from different persons. There was frequent detection of antibodies to a 44-kDa protein of the HGE agent. Serologic testing also revealed possible cases of Lyme borreliosis (
n
= 142), babesiosis (
n
= 41), and HME (
n
= 21). Forty-seven (26.1%) of 180 patients had antibodies to two or more tick-borne agents. Therefore, when one of these diseases is clinically suspected or diagnosed, clinicians should consider the possibility of other current or past tick-borne infections.
PCR for Detection and Identification of
Abiotrophia
spp
Authors: Roggenkamp Andreas , Leitritz Lorenz , Baus Kerstin , Falsen Enevold , Heesemann Jürgen
Published: December 2019

Members of the genus
Abiotrophia
, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of
Abiotrophia
spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of
Abiotrophia
spp. were developed. The first PCR assay was positive for all
Abiotrophia
spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for
A. elegans
, the most fastidious species of
Abiotrophia
. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of
Abiotrophia
spp.
Prolonged Replication of a Type 1 Vaccine-Derived Poliovirus in an Immunodeficient Patient
Authors: Kew Olen M. , Sutter Roland W. , Nottay Baldev K. , McDonough Michael J. , Prevots D. Rebecca , Quick Linda , Pallansch Mark A.
Published: December 2019

VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by ∼10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of ∼3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started ∼9.3 years earlier. This estimate is about the time (6.9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.
Prospective Evaluation of Criteria for Microbiological Diagnosis of Prosthetic-Joint Infection at Revision Arthroplasty
Authors: Atkins Bridget L. , Athanasou Nicholas , Deeks Jonathan J. , Crook Derrick W. M. , Simpson Hamish , Peto Timothy E. A. , McLardy-Smith Peter , Berendt Anthony R. , Group The Osiris Collaborative Study
Published: December 2019

A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic joint infection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to the management and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue were obtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histology as the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using different cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom 297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could not be interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all samples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples taken. The isolation of an indistinguishable microorganism from three or more independent specimens was highly predictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sensitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performance of the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosis of infection be three or more operative specimens that yield an indistinguishable organism, and that because of its low level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.