bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Selection for Mutators in
Escherichia coli
Authors: Miller Jeffrey H. , Suthar Anjali , Tai Jennifer , Yeung Annie , Truong Cindy , Stewart Jean Lee 
Published: December 2019

ABSTRACT

We have constructed strains that allow a direct selection for mutators of
Escherichia coli
on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (
mutHLS
and
uvrD
). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10
−5
in the population. Also, we can measure a mutator cascade, in which one type of mutator (
mutT
) generates a second mutator (
mutHLS
) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.
Complete Sequence of a 184-Kilobase Catabolic Plasmid from
Sphingomonas aromaticivorans
F199
Authors: Romine Margaret F. , Stillwell Lisa C. , Wong Kwong-Kwok , Thurston Sarah J. , Sisk Ellen C. , Sensen Christoph , Gaasterland Terry , Fredrickson Jim K. , Saffer Jeffrey D. 
Published: December 2019

ABSTRACT

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from
Sphingomonas aromaticivorans
F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene,
m
-xylene, and
p
-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another
Sphingomonas
sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.
The P

alkBFGHJKL

Promoter Is under Carbon Catabolite Repression Control in
Pseudomonas oleovorans
but Not in
Escherichia coli alk
+
Recombinants
Authors: Staijen Ivo E. , Marcionelli Rosanna , Witholt Bernard 
Published: December 2019

ABSTRACT

The
alk
genes are located on the OCT plasmid of
Pseudomonas oleovorans
and encode an inducible pathway for the utilization of
n
-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in
P. oleovorans
and
Escherichia coli alk
+
recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The
alk
genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both
E. coli
and pseudomonads.
Pseudomonas putida
GPo12 is a
P. oleovorans
derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant
E. coli
HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the
alkB
gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in
Pseudomonas
and in
E. coli
strains. These results also indicate that P

alkBFGHJKL

, the P

alk

promoter, might be useful in attaining high expression levels of heterologous genes in
E. coli
grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.
Characterization of the Exopolygalacturonate Lyase PelX ofErwinia chrysanthemi3937
Authors: Shevchik Vladimir E. , Kester Harry C. M. , Benen Jacques A. E. , Visser Jaap , Robert-Baudouy Janine , Hugouvieux-Cotte-Pattat Nicole 
Published: December 2019

ABSTRACTErwinia chrysanthemi3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-α-d-galacturonosidase, PehX, have been previously identified in otherE. chrysanthemistrains. Using a genomic bank of a 3937 mutant with the majorpelgenes deleted, we cloned a pectinase gene identified aspelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of anotherE. chrysanthemistrain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL ofE. chrysanthemibut has a N-terminal extension of 324 residues. The transcription ofpelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation ofpelXexpression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved inpelXexpression. ThepelXmutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers orSaintpaulia ionanthaplants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites −2 to +2. PelX and PehX were shown to be localized in the periplasm ofE. chrysanthemi3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.
Identification of Mechanosensitive Ion Channels in the Cytoplasmic Membrane of
Corynebacterium glutamicum
Authors: Ruffert Susanne , Berrier Catherine , Krämer Reinhard , Ghazi Alexandre 
Published: December 2019

ABSTRACT

Patch-clamp experiments performed on membrane fragments of
Corynebacterium glutamicum
fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.
Identification and Characterization of a DeoR-Specific Operator Sequence Essential for Induction of
dra-nupC-pdp
Operon Expression in
Bacillus subtilis
Authors: Zeng Xianmin , Saxild Hans H. 
Published: December 2019

ABSTRACT

The
deoR
gene located just upstream the
dra-nupC-pdp
operon of
Bacillus subtilis
encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional
lacZ
fusions. It was shown that all of the
cis
-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of
dra
. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position −60 to position −43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3′ half of the palindrome and was located between the −10 and −35 regions of the
dra
promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the
dra-nupC-pdp
expression.
Tyrosine Aminotransferase Catalyzes the Final Step of Methionine Recycling in
Klebsiella pneumoniae
Authors: Heilbronn Jacqueline , Wilson Judith , Berger Bradley J. 
Published: December 2019

ABSTRACT

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified from
Klebsiella pneumoniae
and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the
Escherichia coli
and
Salmonella typhimurium
tyrosine aminotransferases (
tyrB
gene products). The structural gene for the tyrosine aminotransferase was cloned from
K. pneumoniae
and expressed in
E. coli
. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from
E. coli
,
S. typhimurium
,
Paracoccus denitrificans
, and
Rhizobium meliloti
, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the
K. pneumoniae
enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.
CspA, CspB, and CspG, Major Cold Shock Proteins of
Escherichia coli
, Are Induced at Low Temperature under Conditions That Completely Block Protein Synthesis
Authors: Etchegaray Jean-Pierre , Inouye Masayori 
Published: December 2019

ABSTRACT

CspA, CspB, and CspG, the major cold shock proteins of
Escherichia coli
, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37°C in the presence of kanamycin (100 μg/ml) or chloramphenicol (200 μg/ml). After 10 min of incubation with the antibiotics at 37°C, cells were cold shocked at 15°C and labeled with [
35
S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of
cspA
and
cspB
mRNAs for the first hour at 15°C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.
Aspartate 205 in the Catalytic Domain of Naphthalene Dioxygenase Is Essential for Activity
Authors: Parales Rebecca E. , Parales Juanito V. , Gibson David T. 
Published: December 2019

ABSTRACTThe naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonassp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571–586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one α subunit and mononuclear iron in the adjacent α subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene tocis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAPand reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.
Cloning of
mnuA
, a Membrane Nuclease Gene of
Mycoplasma pulmonis
, and Analysis of Its Expression in
Escherichia coli
Authors: Jarvill-Taylor Karalee J. , VanDyk Christina , Minion F. Chris 
Published: December 2019

ABSTRACT

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from
Mycoplasma pulmonis
. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in
Escherichia coli
. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated
mnuA
(for “membrane nuclease”). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an
M. pulmonis
membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with
E. coli
confirmed that
mnuA
coded for a nuclease of unknown specificity. Hybridization studies showed that
mnuA
sequences are found in few
Mycoplasma
species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus
Mycoplasma
.
Characterization of the Divergent
sacBK
and
sacAR
Operons, Involved in Sucrose Utilization by
Lactococcus lactis
Authors: Luesink Evert J. , Marugg Joey D. , Kuipers Oscar P. , de Vos Willem M. 
Published: December 2019

ABSTRACT

The divergently transcribed
sacBK
and
sacAR
operons, which are involved in the utilization of sucrose by
Lactococcus lactis
NZ9800, were examined by transcriptional and gene inactivation studies. Northern analyses of RNA isolated from cells grown at the expense of different carbon sources revealed three sucrose-inducible transcripts: one of 3.2 kb containing
sacB
and
sacK
, a second of 3.4 kb containing
sacA
and
sacR
, and a third of 1.8 kb containing only
sacR
. The inactivation of the
sacR
gene by replacement recombination resulted in the constitutive transcription of the
sacBK
and
sacAR
operons in the presence of different carbon sources, indicating that SacR acts as a repressor of transcription.
LB-AUT7
, a Novel Symbiosis-Regulated Gene from an Ectomycorrhizal Fungus,
Laccaria bicolor
, Is Functionally Related to Vesicular Transport and Autophagocytosis
Authors: Kim Sung-Jae , Bernreuther Daniela , Thumm Michael , Podila Gopi K. 
Published: December 2019

ABSTRACT

We have identified
LB-AUT7
, a gene differentially expressed 6 h after ectomycorrhizal interaction between
Laccaria bicolor
and
Pinus resinosa
. LB-Aut7p can functionally complement its
Saccharomyces cerevisiae
homolog, which is involved in the attachment of autophagosomes to microtubules. Our findings suggest the induction of an autophagocytosis-like vesicular transport process during ectomycorrhizal interaction.
Escherichia coli
Outer Membrane Protein TolC Is Involved in Production of the Peptide Antibiotic Microcin J25
Authors: Delgado Mónica A. , Solbiati José O. , Chiuchiolo María J. , Farías Ricardo N. , Salomón Raúl A. 
Published: December 2019

ABSTRACT

A Tn
5
insertion in
tolC
eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The
tolC
mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.
Twelve
pil
Genes Are Required for Biogenesis of the R64 Thin Pilus
Authors: Yoshida Tetsu , Kim Su-Ryang , Komano Teruya 
Published: December 2019

ABSTRACT

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes,
pilI
to -
V
, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594–3603, 1997). In this study, we introduced frameshift mutations into each of the 14
pil
genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes,
pilK
to -
V
, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes,
traBC
, were previously shown to be required for the expression of the
pil
genes. In addition, the
rci
gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes,
traBC
,
pilK
through
pilV
, and
rci
, are essential for R64 thin pilus formation and function.
HmbR, a Hemoglobin-Binding Outer Membrane Protein of
Neisseria meningitidis
, Undergoes Phase Variation
Authors: Richardson Anthony R. , Stojiljkovic Igor 
Published: December 2019

ABSTRACT

Neisseria meningitidis
uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the
hmbR
expression pattern in strains in which the
hpuB
gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some
hpuB
mutants is suggestive of phase variation of
hmbR
. The length of the poly(G) tract starting at position +1164 of
hmbR
absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring the
hmbR
reading frame. Inactivation of
hmbR
in these strains resulted in a loss of Hb utilization, demonstrating that the change in the
hmbR
gene was responsible for the phenotypic switch. The switching rates from
hmbR
phase off to phase on were ∼5 × 10
−4
in four serogroup C strains, 2 × 10
−2
in the serogroup A isolate, and 7 × 10
−6
in the serogroup B isolate.
Stabilization of the Relaxosome and Stimulation of Conjugal Transfer Are Genetically Distinct Functions of the R1162 Protein MobB
Authors: Perwez Tariq , Meyer Richard J. 
Published: December 2019

ABSTRACT

MobB is a small protein encoded by the broad-host-range plasmid R1162 and required for efficient mobilization of its DNA during conjugation. The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the origin of transfer (
oriT
). We have generated in-frame
mobB
deletions that specifically inactivate the stabilizing effect of MobB while still allowing a high rate of transfer. Thus, MobB has two genetically distinct functions in transfer. The effect of another deletion, extending into
mobA
, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain. The
mobB
mutations that specifically affected stability also resulted in poor growth of cells, due to increased transcription from the promoters adjacent to
oriT
. The effects of the mutations could be suppressed not only by full-length MobB provided in
trans
, as expected, but also by additional copies of
oriT
, cloned in pBR322. In addition, in the presence of MobA both the full-length and truncated forms of MobB stimulated recombination between
oriT
-containing plasmids. We propose a model in which MobB regulates expression of plasmid genes by altering the stability of the relaxosome, in a manner that involves the coupling of plasmid molecules.