bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transformation and Expression of a Cloned
fimA
Gene in
Porphyromonas gingivalis
Authors: Takahashi Yusuke , Kato Daisuke , Hamada Nobushiro , Yoshimoto Hisashi , Umemoto Toshio 
Published: March 2020

ABSTRACT

The
Porphyromonas gingivalis
fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene,
fimA
381
, encoding the fimbrial subunit of
P. gingivalis
381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned
fimA
381
gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the
fimA
381
gene into the plasmid vector pYH420 and transformed into the restriction-deficient
P. gingivalis
host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host’s native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the
fimA
381
gene. These results suggested that transformation of
fimA
gene from a different strain of
P. gingivalis
followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.
Characterization of an Immunogenic Glycocalyx on the Surfaces of
Cryptosporidium parvum
Oocysts and Sporozoites
Authors: Nanduri Jayasri , Williams Selvi , Aji Toshiki , Flanigan Timothy P. 
Published: March 2020

ABSTRACT

Ruthenium red staining of
Cryptosporidium parvum
oocysts revealed the presence of a carbohydrate matrix on their outer bilayers that is characteristic of a glycocalyx. Surface labeling of intact oocysts identified material of high molecular weight (>10
6
) that reacted positively with sera from cryptosporidium-infected patients and with immunoglobulin A monoclonal antibodies.
Clonal Expansion of Antigen-Specific CD4 T Cells following Infection withSalmonella typhimuriumIs Similar in Susceptible (Itys) and Resistant (Ityr) BALB/c Mice
Authors: Chen Zong-ming , Jenkins Marc K. 
Published: March 2020

ABSTRACTThe results show that CD4 T cells specific for a recombinant antigen expressed inSalmonella typhimuriumproliferate normally in mice that express the susceptible form of theItygene at early times after infection but do not retain the capacity to produce gamma interferon later in the infection.
Expanded Safety and Immunogenicity of a Bivalent, Oral, Attenuated Cholera Vaccine, CVD 103-HgR Plus CVD 111, in United States Military Personnel Stationed in Panama
Authors: Taylor David N. , Sanchez José L. , Castro José M. , Lebron Carlos , Parrado Carlos M. , Johnson David E. , Tacket Carol O. , Losonsky Genevieve A. , Wasserman Steven S. , Levine Myron M. , Cryz Stanley J. 
Published: March 2020

ABSTRACT

To provide optimum protection against classical and El Tor biotypes of
Vibrio cholerae
O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10
8
CFU plus CVD 111 at 10
7
CFU, (b) CVD 103-HgR at 10
8
CFU plus CVD 111 at 10
6
CFU, (c) CVD 103-HgR alone at 10
8
CFU, (d) CVD 111 alone at 10
7
CFU, or (e) inactivated
Escherichia coli
placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.
The
Staphylococcus aureus
Alternative Sigma Factor ς
B
Controls the Environmental Stress Response but Not Starvation Survival or Pathogenicity in a Mouse Abscess Model
Authors: Chan Pan F. , Foster Simon J. , Ingham Eileen , Clements Mark O. 
Published: March 2020

ABSTRACT

The role of ς
B
, an alternative sigma factor of
Staphylococcus aureus
, has been characterized in response to environmental stress, starvation-survival and recovery, and pathogenicity. ς
B
was mainly expressed during the stationary phase of growth and was repressed by 1 M sodium chloride. A
sigB
insertionally inactivated mutant was created. In stress resistance studies, ς
B
was shown to be involved in recovery from heat shock at 54°C and in acid and hydrogen peroxide resistance but not in resistance to ethanol or osmotic shock. Interestingly,
S. aureus
acquired increased acid resistance when preincubated at a sublethal pH 4 prior to exposure to a lethal pH 2. This acid-adaptive response resulting in tolerance was mediated via
sigB
. However, ς
B
was not vital for the starvation-survival or recovery mechanisms. ς
B
does not have a major role in the expression of the global regulator of virulence determinant biosynthesis, staphylococcal accessory regulator (
sarA
), the production of a number of representative virulence factors, and pathogenicity in a mouse subcutaneous abscess model. However, SarA upregulates
sigB
expression in a growth-phase-dependent manner. Thus, ς
B
expression is linked to the processes controlling virulence determinant production. The role of ς
B
as a major regulator of the stress response, but not of starvation-survival, is discussed.
Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of
Escherichia coli
Authors: Buddelmeijer Nienke , Aarsman Mirjam E. G. , Kolk Arend H. J. , Vicente Miguel , Nanninga Nanne 
Published: March 2020

ABSTRACT

The localization of cell division protein FtsQ in
Escherichia coli
wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in
ftsQ1
(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.
Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme To Transcribe at Temperature Extremes In Vitro
Authors: Rosenheck Sonia , Choder Mordechai 
Published: March 2020

ABSTRACT

Rpb4 is a subunit of
Saccharomyces cerevisiae
RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in
Escherichia coli
prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.
In Vitro Repair of Gaps in Bacteriophage T7 DNA
Authors: Lai Ying-Ta , Masker Warren 
Published: March 2020

ABSTRACT

An in vitro system based upon extracts of
Escherichia coli
infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.
Myxococcus xanthus sasN
Encodes a Regulator That Prevents Developmental Gene Expression during Growth
Authors: Xu Di , Yang Chun , Kaplan Heidi B. 
Published: March 2020

ABSTRACT

Myxococcus xanthus
multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (
sasB5
, -
14
, -
15
, -
16
, and -
17
) can bypass the starvation and high-cell-density requirements for expression of the
4521
developmental reporter gene. These mutants express
4521
at high levels during growth and development in an
asgB
background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the
sasB
locus cloned from the wild-type chromosome restored the SasB
+
phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed
sasN
-null mutation and Ω4521 Tn
5lac
in an otherwise wild-type background expressed
4521
at a high level during growth and development. A similar
sasN
-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type
sasN
gene product is necessary for normal
M. xanthus
fruiting body development and functions as a critical regulator that prevents
4521
expression during growth.
Identification and Characterization of SpcU, a Chaperone Required for Efficient Secretion of the ExoU Cytotoxin
Authors: Finck-Barbançon Viviane , Yahr Timothy L. , Frank Dara W. 
Published: March 2020

ABSTRACT

In recent studies, we have shown that
Pseudomonas aeruginosa
strains that are acutely cytotoxic in vitro damage the lung epithelium in vivo. Genetic analysis indicated that the factor responsible for acute cytotoxicity was controlled by ExsA and therefore was part of the exoenzyme S regulon. The specific virulence determinant responsible for epithelial damage in vivo and cytotoxicity in vitro was subsequently mapped to the
exoU
locus. The present studies are focused on a genetic characterization of the
exoU
locus. Northern blot analyses and complementation experiments indicated that a region downstream of
exoU
was expressed and that the expression of this region corresponded to increased ExoU secretion. DNA sequence analysis of a region downstream of
exoU
identified several potential coding regions. One of these open reading frames, SpcU (specific
Pseudomonas
chaperone for ExoU), encoded a small 15-kDa acidic protein (137 amino acids [pI 4.4]) that possessed a leucine-rich motif associated with the Syc family of cytosolic chaperones for the
Yersinia
Yops. T7 expression analysis and nickel chromatography of histidine-tagged proteins indicated that ExoU and SpcU associated as a noncovalent complex when coexpressed in
Escherichia coli
. The association of ExoU and SpcU required amino acids 3 to 123 of ExoU. In
P. aeruginosa
, ExoU and SpcU are coordinately expressed as an operon that is controlled at the transcriptional level by ExsA.
Characterization of CorR, a Transcriptional Activator Which Is Required for Biosynthesis of the Phytotoxin Coronatine
Authors: Peñaloza-Vázquez Alejandro , Bender Carol L. 
Published: March 2020

ABSTRACT

Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic
Pseudomonas syringae
. The COR biosynthetic gene cluster in
P. syringae
pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes:
corP
,
corS
, and
corR. corS
is thought to function as a histidine protein kinase, whereas
corP
and
corR
show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of
cfl
, a gene located at the 5′ end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a
corR
mutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (
uidA
) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5′ end of the
cfl
upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the
cfl
transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from −704 to −650 with respect to the
cfl
transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region for
cmaA
, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.
Shewanella putrefaciens mtrBEncodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction
Authors: Beliaev Alexander S. , Saffarini Daad A. 
Published: March 2020

ABSTRACTIron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood.Shewanella putrefaciensis a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants ofS. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5inactivation of an open reading frame (ORF) designatedmtrB. mtrBencodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane ofS. putrefaciens. A 5.2-kb DNA fragment that containsmtrBwas isolated and completely sequenced. A second ORF, designatedmtrA, was found directly upstream ofmtrB. The two ORFs appear to be arranged in an operon.mtrAencodes a putative 10-hemec-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB fromEscherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.
Mutational Analysis of the
Rhizobium etli recA
Operator
Authors: Tapias Angels , Barbé Jordi 
Published: March 2020

ABSTRACT

Based upon our earlier studies (A. Tapias, A. R. Fernández de Henestrosa, and J. Barbé, J. Bacteriol. 179:1573–1579, 1997) we hypothesized that the regulatory sequence of the
Rhizobium etli recA
gene was TTGN
11
CAA. However, further detailed analysis of the
R. etli recA
operator described in the present work suggests that it may in fact be GAACN
7
GTAC. This new conclusion is based upon PCR mutagenesis analysis carried out in the
R. etli recA
operator, which indicates that the GAAC and GTAC submotifs found in the sequence GAACN
7
GTAC are required for the maximal stimulation of in vivo transcription and in vitro DNA-protein complex formation. This DNA-protein complex is also detected when the GAACN
7
GTAC wild-type sequence is modified to obtain GAACN
7
GAAC, GTACN
7
GTAC, or GAACN
7
GTTC. The wild-type promoters of the
Rhizobium meliloti
and
Agrobacterium tumefaciens recA
genes, which also contain the GAACN
7
GTAC sequence, compete with the
R. etli recA
promoter for the DNA-protein complex formation but not with mutant derivatives in any of these motifs, indicating that the
R. etli
,
R. meliloti
, and
A. tumefaciens recA
genes present the same regulatory sequence.
Aspartate Transcarbamylase from the Hyperthermophilic Eubacterium
Thermotoga maritima
: Fused Catalytic and Regulatory Polypeptides Form an Allosteric Enzyme
Authors: Chen Pingguo , Van Vliet Françoise , Van De Casteele Mark , Legrain Christianne , Cunin Raymond , Glansdorff Nicolas 
Published: March 2020

ABSTRACT

In the allosteric aspartate transcarbamylase (ATCase) from the hyperthermophilic eubacterium
Thermotoga maritima
, the catalytic and regulatory functions, which in class B ATCases are carried out by specialized polypeptides, are combined on a single type of polypeptide assembled in trimers. The ATCases from
T. maritima
and
Treponema denticola
present intriguing similarities, suggesting horizontal gene transfer.
Molecular Analysis of a Gene Encoding a Cell-Bound Esterase from
Streptomyces chrysomallus
Authors: Berger Rico , Hoffmann Matthias , Keller Ullrich 
Published: March 2020

ABSTRACT

A gene (
estA
) encoding a 42-kDa cell-bound esterase, EstA, was found to be located 75 bp upstream of the cyclophilin A gene (
cypA
) of
Streptomyces chrysomallus
. Western blot analysis revealed the presence of EstA (42 kDa) in cell extracts of
S. chrysomallus
X2 and
Streptomyces lividans
. EstA specifically hydrolyzes short-chain
p
-nitrophenyl esters. EstA formation starts at the end of growth phase, and its activity level remains constant throughout stationary phase. Expression of
estA
from the melanin (
mel
) promoter in plasmid pIJ702 led to a substantial increase of total esterase activity in streptomycetes.
Role of C-Terminal Domains in Surface Attachment of the Fructosyltransferase of
Streptococcus salivarius
ATCC 25975
Authors: Rathsam Catherine , Jacques Nicholas A. 
Published: March 2020

ABSTRACT

The cell-associated β-
d
-fructosyltransferase of
Streptococcus salivarius
, which is devoid of the cell wall anchoring motif, LPXTG, is released on exposure to its substrate, sucrose. Deletions within the C terminus of the enzyme implicated both the hydrophobic and the proline-glycine-serine-threonine-rich wall-associated domain in stabilizing the enzyme on the cell surface.
PRS5, the Fifth Member of the Phosphoribosyl Pyrophosphate Synthetase Gene Family inSaccharomyces cerevisiae, Is Essential for Cell Viability in the Absence of eitherPRS1orPRS3
Authors: Hernando Yolanda , Parr Adrian , Schweizer Michael 
Published: March 2020

ABSTRACTInSaccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-phosphoribosyl-1(α)-pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a phosphoribosyl pyrophosphate binding site. We regard YOL061w as the fifth member of thePRSgene family,PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Δprs5strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion ofPRS1andPRS5orPRS3andPRS5rendered the strains inviable, which implies thatPRS5plays an important role in the maintenance of PRS function inS. cerevisiae.