bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacterial 2,4-Dioxygenases: New Members of the α/β Hydrolase-Fold Superfamily of Enzymes Functionally Related to Serine Hydrolases
Authors: Fischer Frank , Künne Stefan , Fetzner Susanne 
Published: December 2019

ABSTRACT

1
H
-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from
Pseudomonas putida
33/1 and 1
H
-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from
Arthrobacter ilicis
Rü61a catalyze an N-heterocyclic-ring cleavage reaction, generating
N
-formylanthranilate and
N
-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the α/β hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from
Xanthobacter autotrophicus
GJ10 strongly suggested that Qdo and Hod are structurally related to the α/β hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the α/β hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.
A 5′ RNA Stem-Loop Participates in the Transcription Attenuation Mechanism That Controls Expression of the
Bacillus subtilis trpEDCFBA
Operon
Authors: Sudershana Subita , Du Hansen , Mahalanabis Madhumita , Babitzke Paul 
Published: December 2019

ABSTRACT

The
trp
RNA-binding attenuation protein (TRAP) regulates expression of the
Bacillus subtilis trpEDCFBA
operon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent
trp
leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the
trp
structural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5′ end of the
trp
leader transcript. Deletion of this structure resulted in a dramatic increase in expression of a
trpE′-′lacZ
translational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5′ stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5′ stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5′ stem-loop increases the affinity of TRAP for
trp
leader RNA four- to fivefold, suggesting that the 5′ structure interacts with TRAP. In vitro transcription results indicate that this 5′ structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5′ stem-loop was used to demonstrate that formation of the 5′ structure is required for proper attenuation control of this operon.
Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases
Authors: Vorholt Julia A. , Chistoserdova Ludmila , Stolyar Sergei M. , Thauer Rudolf K. , Lidstrom Mary E. 
Published: December 2019

ABSTRACT

The methylotrophic proteobacterium
Methylobacterium extorquens
AM1 possesses tetrahydromethanopterin (H
4
MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO
2
in
M. extorquens
AM1. The distribution of H
4
MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H
4
MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H
4
MPT-dependent activities were also found in autotrophic
Xanthobacter
strains. However, no H
4
MPT-dependent enzyme activities could be detected in other autotrophic α-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H
4
MPT cyclohydrolase (
mch
genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.
Pathways of Assimilative Sulfur Metabolism in
Pseudomonas putida
Authors: Vermeij Paul , Kertesz Michael A. 
Published: December 2019

ABSTRACT

Cysteine and methionine biosynthesis was studied in
Pseudomonas putida
S-313 and
Pseudomonas aeruginosa
PAO1. Both these organisms used direct sulfhydrylation of
O
-succinylhomoserine for the synthesis of methionine but also contained substantial levels of
O
-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine γ-synthase and cystathionine β-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In
P. aeruginosa
, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows
P. aeruginosa
to grow rapidly with methionine as the sole sulfur source.
P. putida
S-313 also grew well with methionine as the sulfur source, but no cystathionine γ-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway,
P. putida
desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine γ-lyase, which was upregulated under these conditions. A transposon mutant of
P. putida
that was defective in the alkanesulfonatase locus (
ssuD
) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in
P. putida
methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.
The C-Terminal Domain of the
Bordetella pertussis
Autotransporter BrkA Forms a Pore in Lipid Bilayer Membranes
Authors: Shannon Jennifer L. , Fernandez Rachel C. 
Published: December 2019

ABSTRACT

BrkA is a 103-kDa outer membrane protein of
Bordetella pertussis
that mediates resistance to antibody-dependent killing by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of the autotransporter family of proteins. Translocation of the N-terminal domain of the protein across the outer membrane is hypothesized to occur through a pore formed by the C-terminal domain. To test this hypothesis, we performed black lipid bilayer experiments with purified recombinant protein. The BrkA C-terminal protein showed an average single-channel conductance of 3.0 nS in 1 M KCl. This result strongly suggests that the C-terminal autotransporter domain of BrkA is indeed capable of forming a pore.
Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from
Saccharomyces cerevisiae
Authors: Hogan Deborah A. , Auchtung Thomas A. , Hausinger Robert P. 
Published: December 2019

ABSTRACT

The
Saccharomyces cerevisiae
open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to
Escherichia coli
taurine/α-ketoglutarate dioxygenase and 27% identity to
Ralstonia eutropha
TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/α-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate. Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources by
S. cerevisiae
, providing strong evidence that YLL057c plays a role in sulfonate catabolism.
Partitioning, Movement, and Positioning of Nucleoids in
Mycoplasma capricolum
Authors: Seto Shintaro , Miyata Makoto 
Published: December 2019

ABSTRACT

The nucleoids in
Mycoplasma capricolum
cells were visualized by phase-combined fluorescence microscopy of DAPI (4′,6-diamidino-2-phenylindole)-stained cells. Most growing cells in a rich medium had one or two nucleoids in a cell, and no anucleate cells were found. The nucleoids were positioned in the center in mononucleoid cells and at one-quarter and three-quarters of the cell length in binucleoid cells. These formations may have the purpose of ensuring delivery of replicated DNA to daughter cells. Internucleoid distances in binucleoid cells correlated with the cell lengths, and the relationship of DNA content to cell length showed that cell length depended on DNA content in binucleoid cells but not in mononucleoid cells. These observations suggest that cell elongation takes place in combination with nucleoid movement. Lipid synthesis was inhibited by transfer of cells to a medium lacking supplementation for lipid synthesis. The transferred cells immediately stopped dividing and elongated while regular spaces were maintained between the nucleoids for 1 h. After 1 h, the cells changed their shapes from rod-like to round, but the proportion of multinucleoid cells increased. Inhibition of protein synthesis by chloramphenicol induced nucleoid condensation and abnormal positioning, although partitioning was not inhibited. These results suggest that nucleoid partitioning does not require lipid or protein synthesis, while regular positioning requires both. When DNA replication was inhibited, the cells formed branches, and the nucleoids were positioned at the branching points. A model for the reproduction process of
M. capricolum
, including nucleoid migration and cell division, is discussed.
Mutational Analysis of the RecJ Exonuclease of
Escherichia coli
: Identification of Phosphoesterase Motifs
Authors: Sutera Vincent A. , Han Eugene S. , Rajman Luis A. , Lovett Susan T. 
Published: December 2019

ABSTRACT

The
recJ
gene, identified in
Escherichia coli
, encodes a Mg
+2
-dependent 5′-to-3′ exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of
Mycoplasma
and
Mycobacterium tuberculosis
. Multiple genes encoding proteins similar to RecJ are found in some eubacteria, including
Bacillus
and
Helicobacter
, and in the archaea. Among this divergent set of sequences, seven conserved motifs emerge. We demonstrate here that amino acids within six of these motifs are essential for both the biochemical and genetic functions of
E. coli
RecJ. These motifs may define interactions with Mg
2+
ions or substrate DNA. A large family of proteins more distantly related to RecJ is present in archaea, eubacteria, and eukaryotes, including a hypothetical protein in the MgPa adhesin operon of
Mycoplasma
, a domain of putative polyA polymerases in
Synechocystis
and
Aquifex
, PRUNE of
Drosophila
, and an exopolyphosphatase (PPX1) of
Saccharomyces cereviseae
. Because these six RecJ motifs are shared between exonucleases and exopolyphosphatases, they may constitute an ancient phosphoesterase domain now found in all kingdoms of life.
Analysis of F Factor TraD Membrane Topology by Use of Gene Fusions and Trypsin-Sensitive Insertions
Authors: Lee Martin H. , Kosuk Nick , Bailey Jeannie , Traxler Beth , Manoil Colin 
Published: December 2019

ABSTRACTThis report describes a procedure for characterizing membrane protein topology which combines the analysis of reporter protein hybrids and trypsin-sensitive 31-amino-acid insertions generated by using transposons ISphoA/in and ISlacZ/in. Studies of the F factor TraD protein imply that the protein takes on a structure with two membrane-spanning sequences and amino and carboxyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins was ambiguous, based on the trypsin cleavage behavior of a 31-residue insertion.
Insertion Mutations in
pilE
Differentially Alter Gonococcal Pilin Antigenic Variation
Authors: Howell-Adams Becky , Seifert H. Steven 
Published: December 2019

ABSTRACT

Pilus antigenic variation in
Neisseria gonorrhoeae
occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (
pilS
) into the expressed pilin gene (
pilE
), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a
pilS
copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called
cys2
. In this study, we show that disruption or displacement of the
pilE cys2
sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in
pilS
, the analogous insertions in
pilE
impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with
pilS
mutations show that the donor
pilS
and recipient
pilE
play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.
4,4′-Diapophytoene Desaturase: Catalytic Properties of an Enzyme from the C
30
Carotenoid Pathway of
Staphylococcus aureus
Authors: Raisig Axel , Sandmann Gerhard 
Published: December 2019

ABSTRACT

Staphylococcus aureus
synthesizes C
30
carotenoids. Their formation involves the introduction of three double bonds, which is catalyzed by a single enzyme. This enzyme, 4,4′-diapophytoene desaturase from
S. aureus
, was overexpressed in
Escherichia coli
and purified in one step by affinity chromatography, and then the protein was characterized with respect to substrate specificity, cofactor requirement, and oligomerization.
Spore Peptidoglycan Structure in a
cwlD dacB
Double Mutant of
Bacillus subtilis
Authors: Popham David L. , Meador-Parton Jennifer , Costello Catherine E. , Setlow Peter 
Published: December 2019

ABSTRACT

Bacillus subtilis cwlD
and
dacB
mutants produce spore peptidoglycan (PG) with increased cross-linking but with little change in spore core hydration compared to the wild type. A
cwlD dacB
double mutant produced spores with a two- to fourfold greater increase in PG cross-linking and novel muropeptides containing glycine residues but no significant changes in spore resistance or core hydration.
Characterization of the Type 8 Capsular Gene Cluster of
Streptococcus pneumoniae
Authors: Muñoz Rosario , Mollerach Marta , López Rubens , García Ernesto 
Published: December 2019

ABSTRACT

The complete nucleotide sequence of the capsular gene cluster (
cap8
) responsible for the biosynthesis of the capsular polysaccharide of
Streptococcus pneumoniae
type 8 has been determined. The
cap8
gene cluster, located between the genes
dexB and aliA
, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the
cap8
genes was sufficient to transform an unencapsulated type 3
S. pneumoniae
strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.
Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in
Saccharomyces cerevisiae
Authors: Silljé H. H. W. , Paalman J. W. G. , ter Schure E. G. , Olsthoorn S. Q. B. , Verkleij A. J. , Boonstra J. , Verrips C. T. 
Published: December 2019

ABSTRACT

Trehalose and glycogen accumulate in
Saccharomyces cerevisiae
when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO
2
production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.
Heat-Induced Synthesis of ς
32
in
Escherichia coli
: Structural and Functional Dissection of
rpoH
mRNA Secondary Structure
Authors: Morita Miyo , Kanemori Masaaki , Yanagi Hideki , Yura Takashi 
Published: December 2019

ABSTRACT

The heat shock response in
Escherichia coli
depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable ς
32
(
rpoH
gene product), which is required for the transcription of heat shock genes. The heat-induced synthesis of ς
32
occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5′ portion (nucleotides −19 to +247) of
rpoH
mRNA in regulation. We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the “downstream box”) and the internal region of
rpoH
mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift.
BglF, the
Escherichia coli
β-Glucoside Permease and Sensor of the
bgl
System: Domain Requirements of the Different Catalytic Activities
Authors: Chen Qing , Amster-Choder Orna 
Published: December 2019

ABSTRACT

The
Escherichia coli
BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of β-glucosides, it phosphorylates BglG, a positive regulator of
bgl
operon transcription, thus inactivating BglG. In the presence of β-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports β-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIA
bgl
and IIB
bgl
, and a membrane-bound domain, IIC
bgl
, which are covalently linked in the order IIBCA
bgl
. Cys-24 in the IIB
bgl
domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBC
bgl
and IICA
bgl
, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBC
bgl
), are required for the sugar-driven reactions, i.e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIB
bgl
alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIB
bgl
and IIC
bgl
which occur upon the interaction of BglF with the sugar.
A Polypeptide with Similarity to Phycocyanin α-Subunit Phycocyanobilin Lyase Involved in Degradation of Phycobilisomes
Authors: Dolganov Nadia , Grossman Arthur R. 
Published: December 2019

ABSTRACT

To optimize the utilization of photosynthate and avoid damage that can result from the absorption of excess excitation energy, photosynthetic organisms must rapidly modify the synthesis and activities of components of the photosynthetic apparatus in response to environmental cues. During nutrient-limited growth, cyanobacteria degrade their light-harvesting complex, the phycobilisome, and dramatically reduce the rate of photosynthetic electron transport. In this report, we describe the isolation and characterization of a cyanobacterial mutant that does not degrade its phycobilisomes during either sulfur or nitrogen limitation and exhibits an increased ratio of phycocyanin to chlorophyll during nutrient-replete growth. The mutant phenotype was complemented by a gene encoding a polypeptide with similarities to polypeptides that catalyze covalent bond formation between linear tetrapyrrole chromophores and subunits of apophycobiliproteins. The complementing gene, designated
nblB
, is expressed at approximately the same level in cells grown in nutrient-replete medium and medium devoid of either sulfur or nitrogen. These results suggest that the NblB polypeptide may be a constitutive part of the machinery that coordinates phycobilisome degradation with environmental conditions.