bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conjugative Mobilization of the Rolling-Circle Plasmid pIP823 from
Listeria monocytogenes
BM4293 among Gram-Positive and Gram-Negative Bacteria
Authors: Charpentier Emmanuelle , Gerbaud Guy , Courvalin Patrice 
Published: December 2019

ABSTRACT

We determined the sequence and genetic organization of plasmid pIP823, which contains the
dfrD
gene;
dfrD
confers high-level trimethoprim resistance to
Listeria monocytogenes
BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The
rep
gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic
Bacillus
sp. and pUB110 from
Staphylococcus aureus
. The
mob
gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included
L. monocytogenes
,
Enterococcus faecalis
,
S. aureus
,
Bacillus subtilis
, and
Escherichia coli
. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between
L. monocytogenes
and
E. faecalis
, between
L. monocytogenes
and
E. coli
, and between strains of
E. coli
, and by the streptococcal conjugative transposon Tn
1545
from
L. monocytogenes
to
E. faecalis
, and from
L. monocytogenes
and
E. faecalis
to
E. coli
. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in
Listeria
spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.
Mapping an Interface of SecY (PrlA) and SecE (PrlG) by Using Synthetic Phenotypes and In Vivo Cross-Linking
Authors: Harris Chris R. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

SecY and SecE are integral cytoplasmic membrane proteins that form an essential part of the protein translocation machinery in
Escherichia coli
. Sites of direct contact between these two proteins have been suggested by the allele-specific synthetic phenotypes exhibited by pairwise combinations of
prlA
and
prlG
signal sequence suppressor mutations in these genes. We have introduced cysteine residues within the first periplasmic loop of SecY and the second periplasmic loop of SecE, at a specific pair of positions identified by this genetic interaction. The expression of the cysteine mutant pair results in a dominant lethal phenotype that requires the presence of DsbA, which catalyzes the formation of disulfide bonds. A reducible SecY-SecE complex is also observed, demonstrating that these amino acids must be sufficiently proximal to form a disulfide bond. The use of cysteine-scanning mutagenesis enabled a second contact site to be discovered. Together, these two points of contact allow the modeling of a limited region of quaternary structure, establishing the first characterized site of interaction between these two proteins. This study proves that actual points of protein-protein contact can be identified by using synthetic phenotypes.
The Physiological Contribution of
Acinetobacter
PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK
Authors: D’Argenio David A. , Segura Ana , Coco Wayne M. , Bünz Patricia V. , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

VanK is the fourth member of the ubiquitous major facilitator superfamily of transport proteins to be identified that, together with PcaK, BenK, and MucK, contributes to aromatic catabolism in
Acinetobacter
sp. strain ADP1. VanK and PcaK have overlapping specificity for
p
-hydroxybenzoate and, most clearly, for protocatechuate: inactivation of both proteins severely impairs growth with protocatechuate, and the activity of either protein alone can mask the phenotype associated with inactivation of its homolog. Furthermore,
vanK pcaK
double-knockout mutants appear completely unable to grow in liquid culture with the hydroaromatic compound quinate, although such cells on plates convert quinate to protocatechuate, which then accumulates extracellularly and is readily visible as purple staining. This provides genetic evidence that quinate is converted to protocatechuate in the periplasm and is in line with the early argument that quinate catabolism should be physically separated from aromatic amino acid biosynthesis in the cytoplasm so as to avoid potential competition for intermediates common to both pathways. Previous studies of aromatic catabolism in
Acinetobacter
have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the β-ketoadipate pathway and preventing the toxic accumulation of carboxymuconate. By using this procedure, strains with a mutation in structural or regulatory genes blocking degradation of vanillate,
p
-hydroxybenzoate, or protocatechuate were selected. In this study, the overlapping specificity of the VanK and PcaK permeases was exploited to directly select strains with a mutation in either
vanK
or
pcaK
. Spontaneous mutations identified in
vanK
include a hot spot for frameshift mutation due to contraction of a G
6
mononucleotide repeat as well as point mutations producing amino acid substitutions useful for analysis of VanK structure and function. Preliminary second-site suppression analysis using transformation-facilitated PCR mutagenesis in one VanK mutant gave results similar to those using LacY, the prototypic member of the major facilitator superfamily, consistent with the two proteins having a similar mechanism of action. The selection for transport mutants described here for
Acinetobacter
may also be applicable to
Pseudomonas putida
, where the PcaK permease has an additional role in chemotaxis.
Early Expression of the Calmodulin Gene, Which Precedes Appressorium Formation in
Magnaporthe grisea
, Is Inhibited by Self-Inhibitors and Requires Surface Attachment
Authors: Liu Zhi-Mei , Kolattukudy Pappachan E. 
Published: December 2019

ABSTRACT

Fungal conidia contain chemicals that inhibit germination and appressorium formation until they are well dispersed in a favorable environment. Recently, such self-inhibitors were found to be present on the conidia of
Magnaporthe grisea
, and plant surface waxes were found to relieve this self-inhibition. To determine whether the self-inhibitors suppress the expression of early genes involved in the germination and differentiation of conidia, the calmodulin gene was chosen as a representative early gene, because it was found to be expressed early in
Colletotrichum gloeosporioides
and
Colletotrichum trifolii
differentiation. After calmodulin cDNA and genomic DNA from
M. grisea
were cloned, the promoter of the calmodulin gene was fused to a reporter gene, that for green fluorescent protein (GFP), and transformed into the
M. grisea
genome. Confocal microscopic examination and quantitation of expression of GFP green fluorescence showed (i) that the expression of the calmodulin gene decreased significantly when self-inhibition of
M. grisea
appressorium formation occurred because of high conidial density or addition of exogenous self-inhibitors and (ii) that the expression level of this gene was restored when self-inhibition was relieved by the addition of plant surface waxes. The increase in fluorescence correlated with the percentage of conidia that formed appressoria. The induction of calmodulin was also confirmed by RNA blotting. Concanavalin A inhibited surface attachment of conidia, GFP expression, and appressorium formation without affecting germination. The high correlation between GFP expression and appressorium formation strongly suggests that calmodulin gene expression and appressorium formation require surface attachment.
Importance of a 5′ Stem-Loop for Longevity of
papA
mRNA in
Escherichia coli
Authors: Bricker Angela L. , Belasco Joel G. 
Published: December 2019

ABSTRACT

High-level expression of the major pilus subunit (PapA) of uropathogenic strains of
Escherichia coli
results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of
papA
mRNA derives in large measure from the protection afforded by its 5′ untranslated region. This
papA
RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the
papA
message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the
papA
5′ untranslated region, as evidenced by the significant acceleration in
papA
mRNA decay that results from its removal.
Analysis of the 5′ Portion of the Type 19A Capsule Locus Identifies Two Classes of
cpsC
,
cpsD
, and
cpsE
Genes in
Streptococcus pneumoniae
Authors: Morona Judy K. , Morona Renato , Paton James C. 
Published: December 2019

ABSTRACT

Analysis of the sequence data obtained from the 5′ portion of the
Streptococcus pneumoniae
type 19A capsular polysaccharide biosynthesis locus (
cps19a
) revealed that the first seven genes are homologous to the first seven genes in the type 19F (
cps19f
) locus. The former genes were designated
cps19aA
to -
G
and were 70 to 90% identical to their
cps19f
counterparts. Southern hybridization analysis of the
cps
loci from various
S. pneumoniae
serotypes with probes specific for the
cps19aC
,
cps19aD
, and
cps19aE
genes indicated a hybridization pattern complementary to that previously reported for
cps19fC
,
cps19fD
, and
cps19fE
. That is, all serotypes tested contained high-stringency homologues of either the
cps19aC
to -
E
genes or the
cps19fC
to -
E
genes, but not both. On this basis
S. pneumoniae cps
loci can be divided into two distinct classes. Long-range PCR was used to amplify the
cps
regions between
cpsB
and
aliA
from a variety of pneumococcal serotypes. Direct sequencing of the 5′ end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of
cpsC
. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.
Salmonella typhimurium
IroN and FepA Proteins Mediate Uptake of Enterobactin but Differ in Their Specificity for Other Siderophores
Authors: Rabsch Wolfgang , Voigt Wolfgang , Reissbrodt Rolf , Tsolis Renée M. , Bäumler Andreas J. 
Published: December 2019

ABSTRACT

Salmonella typhimurium
possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin. To determine whether both receptors have identical substrate specificities,
fepA
and
iroN
mutants and a double mutant were characterized. While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively.
A High Incidence of Prophage Carriage among Natural Isolates of
Streptococcus pneumoniae
Authors: Ramirez Mario , Severina Elena , Tomasz Alexander 
Published: December 2019

ABSTRACT

The majority (591 of 791, or 76%) of
Streptococcus pneumoniae
clinical isolates examined showed the presence of two or more chromosomal
Sma
I fragments that hybridized with the
lytA
-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (
N
-acetylmuramic acid-
l
-alanine amidase). Strains carrying multiple copies of
lytA
homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several
lytA
-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the
lytA
gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the
lytA
-hybridizing fragments in excess of the host
lytA
represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of
lytA
indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.
Modes of Action of Five Different Endopectate Lyases fromErwinia chrysanthemi3937
Authors: Roy Caroline , Kester Harry , Visser Jaap , Shevchik Vladimir , Hugouvieux-Cotte-Pattat Nicole , Robert-Baudouy Jeanine , Benen Jacques 
Published: December 2019

ABSTRACTFive endopectate lyases from the phytopathogenic bacteriumErwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character. PelA, PelI, and PelL released oligogalacturonates of different sizes, whereas PelD and PelB released mostly unsaturated dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer and trimer, except for PelL, which degraded the primary products to unsaturated tetramer and pentamer in addition to unsaturated dimer and trimer. The bond cleavage frequencies on oligogalacturonates revealed differences in the modes of action of these enzymes that were commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI and PelL. For PelA, preferential products were dependent on the sizes of the oligogalacturonates. Whereas PelB and PelD displayed their highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the largest substrate used. The bond cleavage frequencies and reaction rates were used to estimate the number of subsites of each enzyme.
Importance of
cis
Determinants and Nitrogenase Activity in Regulated Stability of the
Klebsiella pneumoniae
Nitrogenase Structural Gene mRNA
Authors: Simon Holly M. , Gosink Mark M. , Roberts Gary P. 
Published: December 2019

ABSTRACT

The
Klebsiella pneumoniae
nitrogen fixation (
nif
) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30°C). Addition of O
2
or fixed nitrogen or temperature increases to 37°C or more result in the dramatic destabilization of the
nif
mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of
nif
transcriptional regulation, was used to define
cis
determinants required for the regulated stability of the 5.2-kb
nifHDKTY
mRNA and to test the model suggested by earlier work that NifA is required in
trans
to stabilize
nif
mRNA under
nif
-derepressing conditions. O
2
regulation of
nifHDKTY
mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of
nifH
, indicating that a site(s) required for the O
2
-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing
nif
mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of
nifY
mutants and overexpression suggests a possible involvement of the protein in this sensing process.
Mutational Analysis of theStreptococcus pneumoniaeBimodular Class A Penicillin-Binding Proteins
Authors: Paik Johanna , Kern Iza , Lurz Rudi , Hakenbeck Regine 
Published: December 2019

ABSTRACTOne group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the threeStreptococcus pneumoniaeclass A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disruptedpbp2agene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors inS. pneumoniae.
Characterization of a
dam
Mutant of
Serratia marcescens
and Nucleotide Sequence of the
dam
Region
Authors: Ostendorf Tammo , Cherepanov Peter , de Vries Johann , Wackernagel Wilfried 
Published: December 2019

ABSTRACT

The DNA of
Serratia marcescens
has N
6
-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from
S. marcescens
Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (
dam
) coding for the adenine methyltransferase (Dam enzyme) of
S. marcescens
was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a
dam-13
::Tn
9
mutant of
Escherichia coli
. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of
E. coli
. The
dam
gene is located between flanking genes which are similar to those found to the sides of the
E. coli dam
gene. The results of complementation studies indicated that like Dam of
E. coli
and unlike Dam of
Vibrio cholerae
, the Dam enzyme of
S. marcescens
plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors.
The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple Bacterium
Rhodobacter capsulatus
Authors: Horken Kempton M. , Tabita F. Robert 
Published: December 2019

ABSTRACT

Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism
Rhodobacter sphaeroides
, the form I RubisCO from
R. capsulatus
is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified the
R. capsulatus
form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to
R. capsulatus
. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes from
R. capsulatus
and
R. sphaeroides
, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of
R. capsulatus
form I RubisCO. The
R. capsulatus
form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO
2
, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO
2
. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO
2
affinity.
Membrane Association and Multimerization of Secreton Component PulC
Authors: Possot Odile M. , Gérard-Vincent Manon , Pugsley Anthony P. 
Published: December 2019

ABSTRACT

The PulC component of the
Klebsiella oxytoca
pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.
Specific Chromosome Alterations in Fluconazole-Resistant Mutants of
Candida albicans
Authors: Perepnikhatka Valentina , Fischer Frank J. , Niimi Masakazu , Baker Rachel A. , Cannon Richard D. , Wang Ying-Kai , Sherman Fred , Rustchenko Elena 
Published: December 2019

ABSTRACT

The exposure of
Candida albicans
to fluconazole resulted in the nondisjunction of two specific chromosomes in 17 drug-resistant mutants, each obtained by an independent mutational event. The chromosomal changes occurred at high frequencies and were related to the duration of the drug exposure. The loss of one homologue of chromosome 4 occurred after incubation on a fluconazole medium for 7 days. A second change, the gain of one copy of chromosome 3, was observed after exposure for 35 or 40 days. We found that the mRNA levels of
ERG11
,
CDR1
,
CDR2
, and
MDR1
, the candidate fluconazole resistance genes, remained either the same or were diminished. The lack of overexpression of putative drug pumps or the drug target indicated that some other mechanism(s) may be operating. The fluconazole resistance phenotype, electrophoretic karyotypes, and transcript levels of mutants were stable after growth for 112 generations in the absence of fluconazole. This is the first report to demonstrate that resistance to fluconazole can be dependent on chromosomal nondisjunction. Furthermore, we suggest that a low-level resistance to fluconazole arising during the early stages of clinical treatment may occur by this mechanism. These results support our earlier hypothesis that changes in
C. albicans
chromosome number is a common means to control a resource of potentially beneficial genes that are related to important cellular functions.