bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Genetic Analysis of Phosphite and Hypophosphite Oxidation by
Pseudomonas stutzeri
WM88
Authors: Metcalf William W. , Wolfe Ralph S. 
Published: December 2019

ABSTRACT

The first molecular and genetic characterization of a biochemical pathway for oxidation of the reduced phosphorus (P) compounds phosphite and hypophosphite is reported. The pathway was identified in
Pseudomonas stutzeri
WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The genes required for oxidation of both compounds by
P. stutzeri
WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in
Escherichia coli
and
Pseudomonas aeruginosa
. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite intermediate. First, plasmid subclones that conferred oxidation of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidation also failed to confer hypophosphite oxidation. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivatives of the cloned genes were made in vitro and recombined onto the chromosome of
P. stutzeri
WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidation deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidation deleted lost only the ability to oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidation phenotypes displayed by the original
P. stutzeri
WM88 isolate. The DNA sequences of the minimal regions implicated in oxidation of each compound were determined. The region required for oxidation of phosphite to phosphate putatively encodes a binding-protein-dependent phosphite transporter, an NAD
+
-dependent phosphite dehydrogenase, and a transcriptional activator of the
lysR
family. The region required for oxidation of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an α-ketoglutarate-dependent hypophosphite dioxygenase. The finding of genes dedicated to oxidation of reduced P compounds provides further evidence that a redox cycle for P may be important in the metabolism of this essential, and often growth-limiting, nutrient.
Domain Analysis of the FliM Protein of
Escherichia coli
Authors: Mathews Michael A. A. , Tang Hua Lucy , Blair David F. 
Published: December 2019

ABSTRACT

The FliM protein of
Escherichia coli
is required for the assembly and function of flagella. Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including FliN, FliG, phosphorylated CheY, other copies of FliM, and possibly MotA and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C-terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the rest of FliM by a segment (residues 237 to 247) that is poorly conserved, tolerates linker insertion, and may be an interdomain linker. Binding to FliG occurs through multiple segments of FliM, some in the C-terminal domain and others in an N-terminal domain of 144 residues. Binding of FliM to CheY and phospho-CheY was complex. In coprecipitation experiments using purified FliM, the protein bound weakly to unphosphorylated CheY and more strongly to phospho-CheY, in agreement with previous reports. By contrast, in experiments using FliM in fresh cell lysates, the protein bound to unphosphorylated CheY about as well as to phospho-CheY. Determinants for binding CheY occur both near the N terminus of FliM, which appears most important for binding to the phosphorylated protein, and in the C-terminal domain, which binds more strongly to unphosphorylated CheY. Several different deletions and linker insertions in FliM enhanced its binding to phospho-CheY in coprecipitation experiments with protein from cell lysates. This suggests that determinants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangement and function of FliM domains in the flagellar motor.
Gigantism in a Bacterium,Epulopiscium fishelsoni, Correlates with Complex Patterns in Arrangement, Quantity, and Segregation of DNA
Authors: Bresler V. , Montgomery W. L. , Fishelson L. , Pollak P. E. 
Published: December 2019

ABSTRACTEpulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2,000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of ∼30 μm to >500 μm. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and “pinching” of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines.
The
Saccharomyces cerevisiae
GATA Factors Dal80p and Deh1p Can Form Homo- and Heterodimeric Complexes
Authors: Svetlov Vladimir V. , Cooper Terrance G. 
Published: December 2019

ABSTRACT

GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in
Saccharomyces cerevisiae
. Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences. A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer. Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini. Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays. We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form. Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers. If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors.
Methylation of FrzCD Defines a Discrete Step in the Developmental Program of
Myxococcus xanthus
Authors: Geng Yongzhi , Yang Zhaomin , Downard John , Zusman David , Shi Wenyuan 
Published: December 2019

ABSTRACT

Myxococcus xanthus
is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The
frz
signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized
asg
,
bsg
,
csg
, and
esg
mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of
M. xanthus
and that social motility mutants are not blocked in these early developmental steps.
DdlN from Vancomycin-Producing
Amycolatopsis orientalis
C329.2 Is a VanA Homologue with
d
-Alanyl-
d
-Lactate Ligase Activity
Authors: Marshall C. Gary , Wright Gerard D. 
Published: December 2019

ABSTRACT

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in
d
-alanyl-
d
-lactate. A key enzyme in this process is a
d
-alanyl-
d
-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide
d
-alanyl-
d
-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism
Amycolatopsis orientalis
C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for
d
-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.
Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present inMycobacterium fortuitumandMycobacterium tuberculosis
Authors: Aínsa José A. , Blokpoel Marian C. J. , Otal Isabel , Young Douglas B. , De Smet Koen A. L. , Martín Carlos 
Published: December 2019

ABSTRACTA recombinant plasmid isolated from aMycobacterium fortuitumgenomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced intoM. smegmatis. Further characterization of this plasmid allowed the identification of theM. fortuitum tapgene. A homologous gene in theM. tuberculosisH37Rv genome has been identified. TheM. tuberculosis tapgene (Rv1258 in the annotated sequence of theM. tuberculosisgenome) was cloned and conferred low-level resistance to tetracycline when introduced intoM. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing thetapgenes were decreased. When tetracycline accumulation experiments were carried out with theM. fortuitum tapgene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organismM. fortuitumand the important pathogenM. tuberculosisare probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.
Characterization of a Peroxide-Resistant Mutant of the Anaerobic Bacterium
Bacteroides fragilis
Authors: Rocha Edson R. , Smith C. Jeffrey 
Published: December 2019

ABSTRACT

A
Bacteroides fragilis
mutant resistant to hydrogen peroxide and alkyl peroxide was isolated by enrichment in increasing concentrations of hydrogen peroxide. The mutant strain was constitutively resistant to 100 mM H
2
O
2
and 5 mM cumene hydroperoxide (15-min exposure). In contrast, the parent strain was protected against <10 mM H
2
O
2
when the peroxide response was induced with a sublethal concentration of H
2
O
2
, and no protection was observed in untreated cells. In addition, catalase activity in the mutant strain was not repressed in anaerobic cultures as reported previously for the parent strain. Comparison of the protein profile of crude extracts of the
B. fragilis
strains revealed that at least three oxidative stress-induced proteins in the parent strain were constitutively expressed in the mutant as detected by nondenaturing polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these overexpressed proteins confirmed the presence of a deregulated catalase (KatB), an alkyl hydroperoxidase reductase subunit C (AhpC), and a Dps/PexB homologue. Northern blot analysis and
katB
::
cat
transcriptional fusion studies revealed that in the mutant,
katB
was deregulated compared to the parent and that
katB
was controlled by a
trans
-acting regulatory mechanism. Moreover, constitutive expression of KatB and of the AhpC and Dps homologues in the H
2
O
2
-resistant mutant suggests that these proteins may share a common oxidative stress transcriptional regulator and may be involved in
B. fragilis
peroxide resistance.
Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences ingutandpapOperons
Authors: van der Woude Marjan , Hale W. Bradley , Low David A. 
Published: December 2019

ABSTRACTMost of the adenine residues in GATC sequences in theEscherichia colichromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) topapDNA was sufficient to protect the two GATC sequences in thepapregulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABDoperon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of thegutABDoperon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated thatgutABDgene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepapGATC-I and GATC-II sites.
Decay of
ermC
mRNA in a Polynucleotide Phosphorylase Mutant of
Bacillus subtilis
Authors: Bechhofer David H. , Wang Wei 
Published: December 2019

ABSTRACT

ermC
mRNA decay was examined in a mutant of
Bacillus subtilis
that has a deleted
pnpA
gene (coding for polynucleotide phosphorylase). 5′-proximal RNA fragments less than 400 nucleotides in length were abundant in the
pnpA
strain but barely detectable in the wild type. On the other hand, the patterns of 3′-proximal RNA fragments were similar in the wild-type and
pnpA
strains. Northern blot analysis with different probes showed that the 5′ end of the decay intermediates was the native
ermC
5′ end. For one prominent
ermC
RNA fragment, in particular, it was shown that formation of its 3′ end was directly related to the presence of a stalled ribosome. 5′-proximal decay intermediates were also detected for transcripts encoded by the
yybF
gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.
Fitness Landscapes for Effects of Shape on Chemotaxis and Other Behaviors of Bacteria
Authors: Dusenbery David B. 
Published: December 2019

ABSTRACT
Data on the shapes of 218 genera of free-floating or free-swimming bacteria reveal groupings around spherical shapes and around rod-like shapes of axial ratio about 3. Motile genera are less likely to be spherical and have larger axial ratios than nonmotile genera. The effects of shape on seven possible components of biological fitness were determined, and actual fitness landscapes in phenotype space are presented. Ellipsoidal shapes were used as models, since their hydrodynamic drag coefficients can be rigorously calculated in the world of low Reynolds number, where bacteria live. Comparing various shapes of the same volume, and assuming that departures from spherical have a cost that varies with the minimum radius of curvature, led to the following conclusions. Spherical shapes have the largest random dispersal by Brownian motion. Increased surface area occurs in oblate ellipsoids (disk-like), which rarely occur. Elongation into prolate ellipsoids (rod-like) reduces sinking speed, and this may explain why some nonmotile genera are rod-like. Elongation also favors swimming efficiency (to a limited extent) and the ability to detect stimulus gradients by any of three mechanisms. By far the largest effect (several hundred-fold) is on temporal detection of stimulus gradients, and this explains why rod-like shapes and this mechanism of chemotaxis are common.
Physical and Genetic Map of the
Pasteurella multocida
A:1 Chromosome
Authors: Hunt Meredith L. , Ruffolo Carmel G. , Rajakumar Kumar , Adler Ben 
Published: December 2019

ABSTRACT

A physical and genetic map of the
Pasteurella multocida
A:1 genome was generated by using the restriction enzymes
Apa
I,
Ceu
I, and
Not
I. The positions of 23 restriction sites and 32 genes, including 5
rrn
operons, were localized on the 2.35-Mbp single circular chromosome. This report presents the first genetic and physical map for this genus.
mmr
, a
Mycobacterium tuberculosis
Gene Conferring Resistance to Small Cationic Dyes and Inhibitors
Authors: De Rossi Edda , Branzoni Manuela , Cantoni Rita , Milano Anna , Riccardi Giovanna , Ciferri Orio 
Published: December 2019

ABSTRACT

The
mmr
gene, cloned from
Mycobacterium tuberculosis
, was shown to confer to
Mycobacterium smegmatis
resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [
3
H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP.
Mutational Analysis of theEscherichia coliK-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression
Authors: Germon Pierre , Clavel Thierry , Vianney Anne , Portalier Raymond , Lazzaroni Jean Claude 
Published: December 2019

ABSTRACTThe Tol-Pal proteins ofEscherichia coliare involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of sometolAmutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.
The
arcABDC
Gene Cluster, Encoding the Arginine Deiminase Pathway of
Bacillus licheniformis
, and Its Activation by the Arginine Repressor ArgR
Authors: Maghnouj Abdelouahid , de Sousa Cabral Tiago Franco , Stalon Victor , Vander Wauven Corinne 
Published: December 2019

ABSTRACT

The arginine deiminase pathway enables
Bacillus licheniformis
to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the
arc
genes encoding the pathway. They appear clustered in an operon-like structure in the order
arcA
(arginine deiminase),
arcB
(ornithine carbamoyltransferase),
arcD
(putative arginine-ornithine antiporter),
arcC
(carbamate kinase). It was found that
B. licheniformis
has an arginine repressor, ArgR, homologous to the
B. subtilis
arginine repressor AhrC. Mutants affected in
argR
were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the
arc
promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.