bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Cloning, Characterization, and Expression of the M Antigen of
Histoplasma capsulatum
Authors: Zancopé-Oliveira Rosely M. , Reiss Errol , Lott Timothy J. , Mayer Leonard W. , Deepe George S. 
Published: March 2020

ABSTRACT

The major diagnostic antigens of
Histoplasma capsulatum
are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to
H. capsulatum
are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic
H. capsulatum
DNA. This fragment was used to screen an
H. capsulatum
genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from
Aspergillus fumigatus
,
Aspergillus niger
, and
Eimericella nidulans
. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of
H. capsulatum
is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.
Vaccination and Protection of Pigs against Pleuropneumonia with a Vaccine Strain of
Actinobacillus pleuropneumoniae
Produced by Site-Specific Mutagenesis of the ApxII Operon
Authors: Prideaux C. T. , Lenghaus C. , Krywult J. , Hodgson A. L. M. 
Published: March 2020

ABSTRACT

The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the induction of protective immunity to
Actinobacillus pleuropneumoniae
reinfection. In the present study, the gene encoding the ApxII-activating protein,
apxIIC
, was insertionally inactivated on the chromosome of a serovar 7 strain, HS93. Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion,
apxIB
and
apxID
, still occurs in this strain. The resulting mutant strain, HS93C
−
Amp
r
, was found to secrete the unactivated toxin. Pigs vaccinated with live HS93C
−
Amp
r
via the intranasal route were protected against a cross-serovar challenge with a virulent serovar 1 strain of
A. pleuropneumoniae
. This is the first reported vaccine strain of
A. pleuropneumoniae
which can be delivered live to pigs and offers cross-serovar protection against porcine pleuropneumonia.
The Mxi-Spa Type III Secretory Pathway ofShigella flexneriRequires an Outer Membrane Lipoprotein, MxiM, for Invasin Translocation
Authors: Schuch Raymond , Maurelli Anthony T. 
Published: March 2020

ABSTRACTInvasion of epithelial cells byShigella flexneriis mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here thatmxiM, part of themxi-spalocus in theS. flexnerivirulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in amxiMnull mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole.
Alkaline Conditions Accelerate Polymorphonuclear Leukocyte Apoptosis In Vitro
Authors: Leblebicioglu Binnaz , Walters John 
Published: March 2020

ABSTRACTApoptosis was monitored in polymorphonuclear leukocytes (PMNs) cultured under mildly acidic, neutral, and alkaline conditions. Within 3 h, 9.0% of the PMNs underwent apoptosis at pH 6.7, as did 12% at pH 7.2, 38% at pH 7.7, and 60% at pH 8.2. Inhibitors of serine proteases, caspase-1, or caspase-3 significantly inhibited PMN apoptosis at pH 8.2, suggesting an involvement by these enzymes.
Characterization of Exochelins of the
Mycobacterium bovis
Type Strain and BCG Substrains
Authors: Gobin Jovana , Wong Diane K. , Gibson Bradford W. , Horwitz Marcus A. 
Published: March 2020

ABSTRACT

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of
Mycobacterium bovis
, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of
M. bovis
, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The
M. bovis
type strain, five substrains of
M. bovis
BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent
Mycobacterium tuberculosis
all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in
M. tuberculosis
, intermediate in
M. bovis
, and smallest in
M. bovis
BCG.
Characterization of
Proteus mirabilis
Precocious Swarming Mutants: Identification of
rsbA
, Encoding a Regulator of Swarming Behavior
Authors: Belas Robert , Schneider Rachel , Melch Michael 
Published: March 2020

ABSTRACT

Proteus mirabilis
swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of
P. mirabilis
. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the
P. mirabilis
genome encoding RsbA (regulator of swarming behavior) and the
P. mirabilis
homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a
rsbA
mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of
rsbA
. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.
The
Bacillus subtilis
Nucleotidyltransferase Is a tRNA CCA-Adding Enzyme
Authors: Raynal Lelia C. , Krisch Henry M. , Carpousis Agamemnon J. 
Published: March 2020

ABSTRACT

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of
Escherichia coli
is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the
E. coli
homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The
Bacillus subtilis papS
gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the
papS
gene should be renamed
cca
, following the notation for its
E. coli
counterpart. The available evidence indicates that
cca
is the only gene encoding an Ntr protein, despite previous suggestions that
B. subtilis
has a PAP similar to
E. coli
PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.
Identification of Conserved, RpoS-Dependent Stationary-Phase Genes of
Escherichia coli
Authors: Schellhorn Herb E. , Audia Jonathon P. , Wei Linda I. C. , Chang Lily 
Published: March 2020

ABSTRACT

During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than 100 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-
lacZ
operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an
rpoS/rpoS
+
merodiploid (
rpoS
background transformed with an
rpoS
-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an
rpoS
background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in
Escherichia coli
do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to
otsA
,
katE
,
ecnB
, and
osmY
—genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely,
gabP
,
yhiUV
,
o371
,
o381
,
f186
, and
o215.
Combined Physical and Genetic Map of the
Pseudomonas putida
KT2440 Chromosome
Authors: Ramos-Díaz M. Angeles , Ramos Juan L. 
Published: March 2020

ABSTRACT

A combined physical and genetic map of the
Pseudomonas putida
KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19
Swa
I, 9
Pme
I, 6
Pac
I, and 6 I-
Ceu
I fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with
Pme
I,
Swa
I, I-
Ceu
I, and
Pac
I as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and
Escherichia coli
, as
P. putida
DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the
P. putida
genome eluted from agarose gels. As an alternative, 10 random mini-Tn
5
-Km mutants of
P. putida
KT2440 were used as a source of DNA, and the band carrying the mini-Tn
5
in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn
5
as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as
oriC
, 6
rrn
loci (
rnnA
to -
F
),
recA
,
ftsZ
,
rpoS
,
rpoD
,
rpoN
, and
gyrB
; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of
P. putida
KT2440 was compared to those of
Pseudomonas aeruginosa
PAO1 and
Pseudomonas fluorescens
SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.
A Two-Component Signal Transduction System Essential for Growth of
Bacillus subtilis
: Implications for Anti-Infective Therapy
Authors: Fabret Céline , Hoch James A. 
Published: March 2020

ABSTRACT

A two-component signal transduction system encoded by the
yycF
and
yycG
genes is part of an operon containing three genes,
yycH
,
yycI
, and
yycJ
, with no known function and a gene,
yycK
, coding for an HtrA-like protease. This operon was transcribed during growth, and its transcription shut down as the cells approached stationary phase. This decreased transcription was not Spo0A dependent. The HtrA protease gene was separately controlled during sporulation from a ς
G
promoter. Studies using insertional inactivation plasmids revealed that neither
yycF
nor
yycG
could be inactivated, whereas the other genes were inactivated without loss of viability. A temperature-sensitive YycF response regulator mutant was isolated and shown to have an H215P mutation in a putative DNA-binding domain which is closely related to the OmpR family of response regulators. At the nonpermissive temperature, cultures of the mutant strain stopped growth within 30 min, and this was followed by a decrease in optical density. Microscopically, many of the cells appeared to retain their structure while being empty of their contents. The essential processes regulated by this two-component system remain unknown. A search of the genome databases revealed YycF, YycG, and YycJ homologues encoded by three linked genes in
Streptococcus pyogenes
. The high level of identity of these proteins (71% for YycF) suggests that this system may play a similar role in gram-positive pathogens.
Sequence Analysis of Tn
10
Insertion Sites in a Collection of
Escherichia coli
Strains Used for Genetic Mapping and Strain Construction
Authors: Nichols Brian P. , Shafiq Obaid , Meiners Victoria 
Published: March 2020

ABSTRACT

The chromosomal insertion sites of Tn
10
-containing
Escherichia coli
strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn
10
positions were determined and matched to the
E. coli
chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots
RhsA
and
RhsB
.
Identification and Characterization of the
Myxococcus xanthus argE
Gene
Authors: Harris Baruch Z. , Singer Mitchell 
Published: March 2020

ABSTRACT

The chromosomal acetylornithine deacetylase (
argE
) gene of
Myxococcus xanthus
was identified via homology to acetylornithine deacetylases from other bacterial species. A mutant carrying a disruption in
argE
was unable to grow on minimal media lacking supplemental arginine and formed fruiting bodies and spores in response to arginine starvation at high cell density.
mraY
Is an Essential Gene for Cell Growth in
Escherichia coli
Authors: Boyle David S. , Donachie William D. 
Published: March 2020

ABSTRACT

The synthesis of the murein precursor lipid I is performed by MraY. We have shown that
mraY
is an essential gene for cell growth. Cells depleted of MraY first swell and then lyse. The expression of
mraY
DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Comparison of Three Commercial Rapid Identification Systems for the Unusual Gram-Positive Cocci
Dolosigranulum pigrum
,
Ignavigranum ruoffiae
, and
Facklamia
Species
Authors: LaClaire Leslye L. , Facklam Richard R. 
Published: March 2020

ABSTRACT

We evaluated three rapid identification systems—The Biomerieux rapid ID 32 STREP (ID32), the BBL Crystal rapid gram-positive identification (Crystal), and the Remel IDS RapID STR (IDS) systems—for their ability to identify 7 strains of
Alloiococcus otitidis
, 27 strains of
Dolosigranulum pigrum
, 3 strains of
Ignavigranum ruoffiae
, and 18 strains of 4 different
Facklamia
species. Since none of these six species of gram-positive cocci are included in the identification databases for these systems, the correct identification for the strains tested should be “unacceptable ID” for the ID32 and Crystal systems or “no choice” for the IDS system. The ID32 system identified all 27 strains of
D. pigrum
, 6 of 18
Facklamia
species, and 2 of 3 cultures of
I. ruoffiae
as “unacceptable ID.” The Crystal system identified 10 of 27
D. pigrum
, 2 of 18
Facklamia
species, and 2 of 3
I. ruoffiae
strains as “unacceptable ID.” The IDS system identified only 1 culture of
D. pigrum
as “no choice,” but it also identified 2 cultures of
D. pigrum
as a “questionable microcode” and 19 cultures of
D. pigrum
as an “inadequate ID,
E. faecalis
90%,
S. intermedius
9%.” A total of 2 of the 18 cultures of
Facklamia
and all 3 of the
I. ruoffiae
cultures were correctly identified as “no choice.” The most common misidentifications of
Facklamia
species by the ID32 and IDS systems were as various
Streptococcus
species and as
Gemella
species. In the Crystal system, the most common erroneous identification was
Micrococcus luteus
. These data indicate the need for the commercial manufacturers of these products to update their databases to include newly described species of gram-positive cocci.
Evaluation of a New System, VITEK 2, for Identification and Antimicrobial Susceptibility Testing of Enterococci
Authors: Garcia-Garrote Fernando , Cercenado Emilia , Bouza Emilio 
Published: March 2020

ABSTRACT

We evaluated the new automated VITEK 2 system (bioMérieux) for the identification and antimicrobial susceptibility testing of enterococci. The results obtained with the VITEK 2 system were compared to those obtained by reference methods: standard identification by the scheme of Facklam and Sahm [R. R. Facklam and D. F. Sahm, p. 308–314,
in
P. R. Murray et al., ed.,
Manual of Clinical Microbiology
, 6th ed., 1995] and with the API 20 STREP system and, for antimicrobial susceptibility testing, broth microdilution and agar dilution methods by the procedures of the National Committee for Clinical Laboratory Standards. The presence of
vanA
and
vanB
genes was determined by PCR. A total of 150 clinical isolates were studied, corresponding to 60
Enterococcus faecalis
, 55
Enterococcus faecium
, 26
Enterococcus gallinarum
, 5
Enterococcus avium
, 2
Enterococcus durans
, and 2
Enterococcus raffinosus
isolates. Among those isolates, 131 (87%) were correctly identified to the species level with the VITEK 2 system. Approximately half of the misidentifications were for
E. faecium
with low-level resistance to vancomycin, identified as
E. gallinarum
or
E. casseliflavus
; however, a motility test solved the discrepancies and increased the agreement to 94%. Among the strains studied, 66% were vancomycin resistant (57 VanA, 16 VanB, and 26 VanC strains), 23% were ampicillin resistant (MICs, ≥16 μg/ml), 31% were high-level gentamicin resistant, and 45% were high-level streptomycin resistant. Percentages of agreement for susceptibility and resistance to ampicillin, vancomycin, and teicoplanin and for high-level gentamicin resistance and high-level streptomycin resistance were 93, 95, 97, 97, and 96%, respectively. The accuracy of identification and antimicrobial susceptibility testing of enterococci with the VITEK 2 system, together with the significant reduction in handling time, will have a positive impact on the work flow of the clinical microbiology laboratory.
Genetic Diversity of
Borrelia burgdorferi
Sensu Lato in Ticks from Mainland Portugal
Authors: De Michelis Simona , Sewell Henna-Sisko , Collares-Pereira Margarida , Santos-Reis Margarida , Schouls Leo M. , Benes Vladimir , Holmes Edward C. , Kurtenbach Klaus 
Published: March 2020

ABSTRACT

To date
Borrelia lusitaniae
is the only genospecies of
Borrelia burgdorferi
sensu lato isolated from
Ixodes ricinus
ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of
B. burgdorferi
sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of
B. burgdorferi
sensu lato from this region, 55
I. ricinus
and 27
Hyalomma marginatum
questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in
I. ricinus
ticks and 7% in
H. marginatum
ticks were detected by a nested PCR that targets the
rrf
(5S)-
rrl
(23S) spacer of
B. burgdorferi
sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the
I. ricinus
-derived amplicons showed that the sequences in the majority of samples were similar to those of
B. lusitaniae
type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of
rrf-rrl
spacer sequences revealed a diverse population of
B. lusitaniae
in questing adult
I. ricinus
ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of
B. lusitaniae
were present in the local
I. ricinus
population. We hypothesize that
B. lusitaniae
has a narrow ecological niche that involves host species restricted to the Mediterranean Basin.