bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spent Culture Supernatant of
Mycobacterium tuberculosis
H37Ra Improves Viability of Aged Cultures of This Strain and Allows Small Inocula To Initiate Growth
Authors: Sun Zhonghe , Zhang Ying 
Published: December 2019

ABSTRACT

Spent culture supernatant from early-stationary-phase
Mycobacterium tuberculosis
H37Ra cultures increased the viability of bacilli from aged cultures of this strain and allowed small inocula to initiate growth in liquid culture. The resuscitation factor was acid labile and heat stable, with a mass of less than 1,375 Da.
IS
6110
-Mediated Deletions of Wild-Type Chromosomes of
Mycobacterium tuberculosis
Authors: Fang Z. , Doig C. , Kenna D. T. , Smittipat N. , Palittapongarnpim P. , Watt B. , Forbes K. J. 
Published: December 2019

ABSTRACT

The
ipl
locus is a site for the preferential insertion of IS
6110
and has been identified as an insertion sequence, IS
1547
, in its own right. Various deletions around the
ipl
locus of clinical isolates of
Mycobacterium tuberculosis
were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS
6110
copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS
6110
transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS
6110
was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of
M. tuberculosis
, for IS
6110
-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of
M. tuberculosis
has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.
The
udhA
Gene of
Escherichia coli
Encodes a Soluble Pyridine Nucleotide Transhydrogenase
Authors: Boonstra Birgitte , French Christopher E. , Wainwright Ian , Bruce Neil C. 
Published: December 2019

ABSTRACT

The
udhA
gene of
Escherichia coli
was cloned and expressed in
E. coli
and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published
E. coli
genome sequences due to a sequencing error.
E. coli
is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.
Analysis of FtsZ Assembly by Light Scattering and Determination of the Role of Divalent Metal Cations
Authors: Mukherjee Amit , Lutkenhaus Joe 
Published: December 2019

ABSTRACT

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90° angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg
2+
was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca
2+
mediated dynamic assembly of FtsZ (X.-C. Yu and W. Margolin, EMBO J. 16:5455–5463, 1997). However, we found that Ca
2+
was not required for FtsZ assembly and that this concentration of Ca
2+
reduced the dynamic behavior of FtsZ assembly.
The SPI-3 Pathogenicity Island of
Salmonella enterica
Authors: Blanc-Potard Anne-Béatrice , Solomon Felix , Kayser Jayson , Groisman Eduardo A. 
Published: December 2019

ABSTRACT

Pathogenicity islands are chromosomal clusters of pathogen-specific virulence genes often found at tRNA loci. We have determined the molecular genetic structure of SPI-3, a 17-kb pathogenicity island located at the
selC
tRNA locus of
Salmonella enterica
serovar Typhimurium. The G+C content of SPI-3 (47.5%) differs from that of the
Salmonella
genome (52%), consistent with the notion that these sequences have been horizontally acquired. SPI-3 harbors 10 open reading frames organized in six transcriptional units, which include the previously described
mgtCB
operon encoding the macrophage survival protein MgtC and the Mg
2+
transporter MgtB. Among the newly identified open reading frames, one exhibits sequence similarity to the ToxR regulatory protein of
Vibrio cholerae
and one is similar to the AIDA-I adhesin of enteropathogenic
Escherichia coli
. The distribution of SPI-3 sequences varies among the salmonellae: the right end of the island, which harbors the virulence gene
mgtC
, is present in all eight subspecies of
Salmonella
; however, a four-gene cluster at the center of SPI-3 is found in only some of the subspecies and is bracketed by remnants of insertion sequences, suggesting a multistep process in the evolution of SPI-3 sequences.
Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids
Authors: Takahashi Shunji , Kuzuyama Tomohisa , Seto Haruo 
Published: December 2019

ABSTRACT

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC
1.1.1.34
) was purified 3,000-fold from
Streptomyces
sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH
4
)
2
SO
4
precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent

K
m

values of 62 μM for NADPH and 7.7 μM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.
Cloning and Characterization of Arylamine N -Acetyltransferase Genes from
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
: Increased Expression Results in Isoniazid Resistance
Authors: Payton Mark , Auty Roy , Delgoda Rupika , Everett Martin , Sim Edith 
Published: December 2019

ABSTRACT

Arylamine
N
-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote
Salmonella typhimurium
. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it.
nat
genes were cloned from
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
, and expressed in
Escherichia coli
and
M. smegmatis
. The induced
M. smegmatis
NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from
S. typhimurium
recognizes NAT from
M. smegmatis
and cross-reacts with recombinant NAT from
M. tuberculosis
. Overexpression of mycobacterial
nat
genes in
E. coli
results in predominantly insoluble recombinant protein; however, with
M. smegmatis
as the host using the vector pACE-1, NAT proteins from
M. tuberculosis
and
M. smegmatis
are soluble.
M. smegmatis
transformants induced to express the
M. tuberculosis nat
gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.
Chromosomal Changes during Experimental Evolution in Laboratory Populations of
Escherichia coli
Authors: Bergthorsson Ulfar , Ochman Howard 
Published: December 2019

ABSTRACT

Short-term rates of chromosome evolution were analyzed in experimental populations of
Escherichia coli
B that had been propagated for 2,000 generations under four thermal regimens. Chromosome alterations were monitored in 24 independent populations by pulsed-field gel electrophoresis of DNA treated with five rare-cutting restriction enzymes. A total of 11 changes, 8 affecting chromosome size and 3 altering restriction sites, were observed in these populations, with none occurring in strains cultured at 37°C. Considering the changes detected in these experimental populations, the rate of chromosome alteration of
E. coli
is estimated to be half of that observed in experimental populations of yeast.
Lack of Genetic Differentiation between Two Geographically Diverse Samples of
Candida albicans
Isolated from Patients Infected with Human Immunodeficiency Virus
Authors: Xu Jianping , Vilgalys Rytas , Mitchell Thomas G. 
Published: December 2019

ABSTRACT

The patterns of genetic variation of samples of
Candida albicans
isolated from patients infected with human immunodeficiency virus in Durham, N.C., and Vitória, Brazil, were compared. Genotypes for 126 strains were obtained at 16 polymorphic restriction sites distributed on nine PCR fragments. The results indicated evidence of clonality both within and between these two geographically diverse samples. The samples are genetically very similar, with little evidence of genetic differentiation.
Ferritin Mutants of
Escherichia coli
Are Iron Deficient and Growth Impaired, and
fur
Mutants are Iron Deficient
Authors: Abdul-Tehrani Hossein , Hudson Aaron J. , Chang Yung-Sheng , Timms Andrew R. , Hawkins Chris , Williams John M. , Harrison Pauline M. , Guest John R. , Andrews Simon C. 
Published: December 2019

ABSTRACT

Escherichia coli
contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (
ftnA
and
bfr
) were inactivated by replacing the chromosomal
ftnA
and
bfr
genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (
ftnA::spc
and
bfr::kan
) and a double mutant (
ftnA::spc bfr::kan
) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the
bfr
mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the
ftnA
and
ftnA bfr
mutants remained unchanged. The
ftnA
and
ftnA bfr
mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (
fur
) mutants also had very low iron contents (2.5-fold less iron than Fur
+
strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the
ftnA
and
fur
mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of
ftnA
mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the
fur
mutation,
ftnA
and
bfr
mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.