bluuebunny/crossref_metadata_2025_split huggingface dataset:
The A Modules of the
Azotobacter vinelandii
Mannuronan-C-5-Epimerase AlgE1 Are Sufficient for both Epimerization and Binding of Ca
2+
Authors: Ertesvåg Helga , Valla Svein 
Published: December 2019

ABSTRACT

The industrially important polysaccharide alginate is composed of the two sugar monomers β-
d
-mannuronic acid (M) and its epimer α-
l
-guluronic acid (G). In the bacterium
Azotobacter vinelandii
, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A
1
R
1
R
2
R
3
A
2
R
4
. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A
1
catalyzed the formation of contiguous stretches of G residues in the polymer, while A
2
introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca
2+
dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca
2+
concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.
Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in
Vibrio cholerae
Authors: Sandkvist Maria , Hough Lloyd P. , Bagdasarian Mira M. , Bagdasarian Michael 
Published: December 2019

ABSTRACT

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM of
Vibrio cholerae
, have been purified and characterized. Based on gel filtration analysis, both purified EpsM

(His)
6

and wild-type EpsL present in an
Escherichia coli
Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in
E. coli
, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.
Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol fromPseudomonas fluorescensQ2-87
Authors: Bangera M. Gita , Thomashow Linda S. 
Published: December 2019

ABSTRACTThe polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescentPseudomonasspp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis byP. fluorescensQ2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipientPseudomonasstrains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side byphlEandphlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression inEscherichia coliofphlA,phlC,phlB, andphlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.
Enhanced Function Conferred on Low-Abundance Chemoreceptor Trg by a Methyltransferase-Docking Site
Authors: Feng Xiuhong , Lilly Angela A. , Hazelbauer Gerald L. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, high-abundance chemoreceptors are present in cellular amounts approximately 10-fold higher than those of low-abundance receptors. These two classes exhibit inherent differences in functional activity. As sole cellular chemoreceptors, high-abundance receptors are effective in methyl-accepting activity, in establishing a functional balance between the two directions of flagellar rotation, in timely adaptation, and in mediating efficient chemotaxis. Low-abundance receptors are not, even when their cellular content is increased. We found that the low-abundance receptor Trg acquired essential functional features of a high-abundance receptor by the addition of the final 19 residues of the high-abundance receptor Tsr. The carboxy terminus of this addition carried a methyltransferase-binding pentapeptide, NWETF, present in high-abundance receptors but absent in the low-abundance class. Provision of this docking site not only enhanced steady-state and adaptational methylation but also shifted the abnormal, counterclockwise bias of flagellar rotation toward a more normal rotational balance and vastly improved chemotaxis in spatial gradients. These improvements can be understood as the result of both enhanced kinase activation by the more methylated receptor and timely adaptation by more efficient methyl-accepting activity. We conclude that the crucial functional difference between the low-abundance receptor Trg and its high-abundance counterparts is the level of methyl-accepting activity conferred by the methyltransferase-docking site.
Sodium-Dependent Glutamate Uptake by an Alkaliphilic, Thermophilic
Bacillus
Strain, TA2.A1
Authors: Peddie Catherine J. , Cook Gregory M. , Morgan Hugh W. 
Published: December 2019

ABSTRACT

A strain of
Bacillus
designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C.
Bacillus
strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (ΔpNa) across the cell membrane.
N
,
N
-Dicyclohexylcarbodiimide inhibited the growth of
Bacillus
strain TA2.A1, suggesting that an F
1
F
0
-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of
Bacillus
strain TA2.A1. Glutamate transport by
Bacillus
strain TA2.A1 could be driven by an artificial membrane potential (ΔΨ), but only when sodium was present. In the absence of sodium, the rate of ΔΨ-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of ΔpNa alone (i.e., no ΔΨ). Glutamate uptake was specifically inhibited by monensin, and the

K
m

for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide
m
-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the

K
m

for glutamate was 2.90 μM, and the
V
max
was 0.7 nmol · min
−1
mg of protein. Glutamate transport was specifically inhibited by glutamate analogues.
Amino Acid Biosynthesis in the Halophilic Archaeon
Haloarcula hispanica
Authors: Hochuli Michel , Patzelt Heiko , Oesterhelt Dieter , Wüthrich Kurt , Szyperski Thomas 
Published: December 2019

ABSTRACT

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon
Haloarcula hispanica
was explored by using biosynthetically directed fractional
13
C labeling with a mixture of 90% unlabeled and 10% uniformly
13
C-labeled glycerol. The resulting
13
C-labeling patterns in the amino acids were analyzed by two-dimensional
13
C,
1
H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the
13
C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.
Purification, Kinetic Properties, and Intracellular Concentration of SpoIIE, an Integral Membrane Protein That Regulates Sporulation in
Bacillus subtilis
Authors: Lucet Isabelle , Borriss Rainer , Yudkin Michael D. 
Published: December 2019

ABSTRACT

SpoIIE is a bifunctional protein which controls ς
F
activation and formation of the asymmetric septum in sporulating
Bacillus subtilis
. The
spoIIE
gene of
B. subtilis
has now been overexpressed in
Escherichia coli
, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.
Carbon and Electron Flow in
Clostridium cellulolyticum
Grown in Chemostat Culture on Synthetic Medium
Authors: Guedon E. , Payot S. , Desvaux M. , Petitdemange H. 
Published: December 2019

ABSTRACT

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when
Clostridium cellulolyticum
was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD
+
ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h
−1
) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.
Three Surface Layer Homology Domains at the N Terminus of the
Clostridium cellulovorans
Major Cellulosomal Subunit EngE
Authors: Tamaru Yutaka , Doi Roy H. 
Published: December 2019

ABSTRACT

The gene
engE
, coding for endoglucanase E, one of the three major subunits of the
Clostridium cellulovorans
cellulosome, has been isolated and sequenced.
engE
is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796. The amino acid sequence derived from
engE
revealed a structure consisting of catalytic and noncatalytic domains. The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium
Caulobacter crescentus
. The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus.
engE
is located downstream of an ORF, ORF1, that is homologous to the
Bacillus subtilis
phosphomethylpyrimidine kinase (
pmk
) gene. The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of
C. cellulovorans
.
Identification and In Vivo Functional Analysis of a Virginiamycin S Resistance Gene (
varS
) from
Streptomyces virginiae
Authors: Lee Chang-Kwon , Kamitani Yuka , Nihira Takuya , Yamada Yasuhiro 
Published: December 2019

ABSTRACT

BarA of
Streptomyces virginiae
is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the
Streptomyces
species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis. The downstream gene
barB
, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the
barB
termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named
varS
(for
S. virginiae
antibiotic resistance). Heterologous expression of
varS
with
S. lividans
as a host resulted in virginiamycin S-specific resistance, suggesting that
varS
encoded a virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of
barB-varS
appeared 1 to 2 h before the onset of virginiamycin M
1
and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic
varS
transcript.
Mutant Forms of
Salmonella typhimurium
ς
54
Defective in Transcription Initiation but Not Promoter Binding Activity
Authors: Kelly Mary T. , Hoover Timothy R. 
Published: December 2019

ABSTRACT

Transcription initiation with ς
54
-RNA polymerase holoenzyme (ς
54
-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς
54
’s binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of
Salmonella typhimurium
ς
54
that were deficient in transcription initiation but still directed ς
54
-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for ς
54
-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς
54
at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.
Cloning and Characterization of the Genes Encoding a Cytochrome P450 (PipA) Involved in Piperidine and Pyrrolidine Utilization and Its Regulatory Protein (PipR) in
Mycobacterium smegmatis
mc
2
155
Authors: Poupin Pascal , Ducrocq Véronique , Hallier-Soulier Sylvie , Truffaut Nicole 
Published: December 2019

ABSTRACT

Transposon mutagenesis of
Mycobacterium smegmatis
mc
2
155 enabled the isolation of a mutant strain (called LGM1) altered in the regulation of piperidine and pyrrolidine utilization. The complete nucleotide sequence of the gene inactivated in mutant LGM1 was determined from the wild-type strain. This gene (
pipR
) encoded a member of the GntR family of bacterial regulatory proteins. An insertion element (IS
1096
), previously described for
M. smegmatis
, was detected downstream of the gene
pipR
. Three additional open reading frames were found downstream of IS
1096
. The first open reading frame (
pipA
) appeared to encode a protein identified as a cytochrome P450 enzyme. This gene is the first member of a new family,
CYP151
. By a gene replacement experiment, it was demonstrated that the cytochrome P450
pipA
gene is required for piperidine and pyrrolidine utilization in
M. smegmatis
mc
2
155. Genes homologous to
pipA
were detected by hybridization in several, previously isolated, morpholine-degrading mycobacterial strains. A gene encoding a putative [3Fe-4S] ferredoxin (
orf1
) and a truncated gene encoding a putative glutamine synthetase (
orf2
′) were found downstream of
pipA
.
Organization and Differential Regulation of a Cluster of Lignin Peroxidase Genes of
Phanerochaete chrysosporium
Authors: Stewart Philip , Cullen Daniel 
Published: December 2019

ABSTRACT

The lignin peroxidases of
Phanerochaete chrysosporium
are encoded by a minimum of 10 closely related genes. Physical and genetic mapping of a cluster of eight
lip
genes revealed six genes occurring in pairs and transcriptionally convergent, suggesting that portions of the
lip
family arose by gene duplication events. The completed sequence of
lipG
and
lipJ
, together with previously published sequences, allowed phylogenetic and intron/exon classifications, indicating two main branches within the
lip
family. Competitive reverse transcription-PCR was used to assess
lip
transcript levels in both carbon- and nitrogen-limited media. Transcript patterns showed differential regulation of
lip
genes in response to medium composition. No apparent correlation was observed between genomic organization and transcript levels. Both constitutive and upregulated transcripts, structurally unrelated to peroxidases, were identified within the
lip
cluster.
Cd(II)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR
Authors: Caguiat Jonathan J. , Watson Alice L. , Summers Anne O. 
Published: December 2019

ABSTRACT

Expression of the Tn
21
mercury resistance (
mer
) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator
merO
), repressing transcription of the structural genes
merTPCAD
in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the
merO
DNA to facilitate transcriptional initiation by ς
70
RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought
merR
mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR’s metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.
Cloning of the Glutamyl-tRNA Synthetase (
gltX
) Gene from
Pseudomonas aeruginosa
Authors: Franklund Clifton V. , Goldberg Joanna B. 
Published: December 2019

ABSTRACT

The glutamyl-tRNA synthetase (
gltX
) gene from
Pseudomonas aeruginosa
was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive
gltX
mutation of
Escherichia coli
JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from
Rhizobium meliloti
.