bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppression of TGA Mutations in the
Bacillus subtilis spoIIR
Gene by
prfB
Mutations
Authors: Karow Margaret L. , Rogers Elizabeth J. , Lovett Paul S. , Piggot Patrick J. 
Published: December 2019

ABSTRACT

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the
spoIIR
locus of
Bacillus subtilis
. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the
prfB
gene encoding protein release factor 2. These are the first mutations shown to map to the
B. subtilis prfB
locus. The sequence of the
prfB
gene was completed, and two revisions of the published sequence were made. The five
prfB
mutations also resulted in suppression of the
catA86
-TGA mutation to between 19 and 54% of the expression of
catA86
+
, compared to the readthrough level of 6% in the
prfB
+
strain. N-terminal sequencing of suppressed
catA86
-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the
prfB1
mutations was tryptophan.
Physiological Control and Regulation of the
Rhodobacter capsulatus cbb
Operons
Authors: Paoli George C. , Vichivanives Padungsri , Tabita F. Robert 
Published: December 2019

ABSTRACT

The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in
Rhodobacter capsulatus
are organized in at least two operons, each preceded by a separate
cbbR
gene, encoding potential LysR-type transcriptional activators. As a prelude to studies of
cbb
gene regulation in
R. capsulatus
, the nucleotide sequence of a 4,537-bp region, which included
cbbR
II
, was determined. This region contained the following open reading frames: a partial
pgm
gene (encoding phosphoglucomutase) and a complete
qor
gene (encoding NADPH:quinone oxidoreductase), followed by
cbbR
II
,
cbbF
(encoding fructose 1,6-bisphosphatase),
cbbP
(encoding phosphoribulokinase), and part of
cbbT
(encoding transketolase). Physiological control of the CBB pathway and regulation of the
R. capsulatus cbb
genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the
cbbLS
and
cbbM
genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both
cbbL
and
cbbM
could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption of
cbbP
resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one
cbbP
gene in
R. capsulatus
and that this gene is cotranscribed with
cbbM
. Analysis of RubisCO activity and synthesis in strains with disruptions in either
cbbR
I
or
cbbR
II
, and β-galactosidase determinations from wild-type and mutant strains containing
cbb
Ip
- and
cbb
IIp
-
lacZ
fusion constructs, indicated that the
cbb
I
and
cbb
II
operons of
R. capsulatus
are within separate CbbR regulons.
Three
cdg
Operons Control Cellular Turnover of Cyclic Di-GMP in
Acetobacter xylinum
: Genetic Organization and Occurrence of Conserved Domains in Isoenzymes
Authors: Tal Rony , Wong Hing C. , Calhoon Roger , Gelfand David , Fear Anna Lisa , Volman Gail , Mayer Raphael , Ross Peter , Amikam Dorit , Weinhouse Haim , Cohen Avital , Sapir Shai , Ohana Patricia , Benziman Moshe 
Published: December 2019

ABSTRACT

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of β-1,4-glucan (cellulose) synthase in
Acetobacter xylinum
. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate,
cdg1
,
cdg2
, and
cdg3
. Within each
cdg
operon, a
pdeA
gene lies upstream of a
dgc
gene.
cdg1
contains two additional flanking genes,
cdg1a
and
cdg1d. cdg1a
encodes a putative transcriptional activator, similar to AadR of
Rhodopseudomonas palustris
and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the
cdg
operons, such that
cdg1
contributes 80% of cellular DGC and PDEA activities and
cdg2
and
cdg3
contribute 15 and 5%, respectively. Disruption of
dgc
genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.
Biochemistry and Regulation of a Novel
Escherichia coli
K-12 Porin Protein, OmpG, Which Produces Unusually Large Channels
Authors: Fajardo Daniel A. , Cheung Joyce , Ito Chikako , Sugawara Etsuko , Nikaido Hiroshi , Misra Rajeev 
Published: December 2019

ABSTRACT

A novel porin, OmpG, is produced in response to a chromosomal mutation termed
cog-192
. Molecular characterization of
cog-192
revealed that it is a large chromosomal deletion extending from the 3′ end of
pspA
through to the 5′ end of an open reading frame located immediately upstream of
ompG
. As a result of this 13.1-kb deletion, the expression of
ompG
was placed under the control of the
pspA
promoter. Characterization of OmpG revealed that it is quite different from other porins. Proteoliposome swelling assays showed that OmpG channels were much larger than those of the OmpF and OmpC porins, with an estimated limited diameter of about 2 nm. The channel lacked any obvious solute specificity. The folding model of OmpG suggests that it is the first 16-stranded β-barrel porin that lacks the large external loop, L3, which constricts the channels of other nonspecific and specific porins. Consistent with the folding model, circular dichroism showed that OmpG contains largely a β-sheet structure. In contrast to other
Escherichia coli
porins, there is no evidence that OmpG exists as stable oligomers. Although
ompG
DNA was present in all
E. coli
strains examined so far, its expression under laboratory conditions was seen only due to rare chromosomal mutations. Curiously, OmpG was constitutively expressed, albeit at low levels, in
Salmonella
,
Shigella
, and
Pseudomonas
species.
Pathway Choice in Glutamate Synthesis in
Escherichia coli
Authors: Helling Robert B. 
Published: December 2019

ABSTRACT

Escherichia coli
has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.
Two-Step Autocatalytic Processing of the Glutaryl 7-Aminocephalosporanic Acid Acylase from
Pseudomonas
sp. Strain GK16
Authors: Lee Young Sik , Park Sung Soo 
Published: December 2019

ABSTRACT

The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of
Pseudomonas
sp. strain GK16 is an (αβ)
2
heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into α and β subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the α subunit and to show that Ser199 of the β subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the α subunit, containing the spacer peptide, and the β subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the β subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198).
Expression of
glnB
and a
glnB
-Like Gene (
glnK
) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of
Rhodobacter sphaeroides
Authors: Qian Yilei , Tabita F. Robert 
Published: December 2019

ABSTRACT

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of
Rhodobacter sphaeroides
, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a
glnB
::
lacZ
fusion, since the product of the
glnB
gene is known to have a negative effect on ammonia-regulated
nif
control. It was found that
glnB
expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by
trans
complementation of the deleted genes, wild-type levels of GS and
glnB
expression were restored. Furthermore, a
glnB
-like gene,
glnK
, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly,
glnK
expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.
Evolution of the Major Pilus Gene Cluster of
Haemophilus influenzae
Authors: Mhlanga-Mutangadura Tendai , Morlin Gregory , Smith Arnold L. , Eisenstark Abraham , Golomb Miriam 
Published: December 2019

ABSTRACT

Haemophilus influenzae
is a ubiquitous colonizer of the human respiratory tract and causes diseases ranging from otitis media to meningitis. Many
H. influenzae
isolates express pili (fimbriae), which mediate adherence to epithelial cells and facilitate colonization. The pilus gene (
hif
) cluster of
H. influenzae
type b maps between
purE
and
pepN
and resembles a pathogenicity island: it is present in invasive strains, absent from the nonpathogenic Rd strain, and flanked by direct repeats of sequence at the insertion site. To investigate the evolution and role in pathogenesis of the
hif
cluster, we compared the
purE-pepN
regions of various
H. influenzae
laboratory strains and clinical isolates. Unlike Rd, most strains had an insert at this site, which usually was the only chromosomal locus of
hif
DNA. The inserts are diverse in length and organization: among 20 strains, nine different arrangements were found. Several nontypeable isolates lack
hif
genes but have two conserved open reading frames (
hicA
and
hicB
) upstream of
purE
; their inferred products are small proteins with no data bank homologs. Other isolates have
hif
genes but lack
hic
DNA or have combinations of
hif
and
hic
genes. By comparing these arrangements, we have reconstructed a hypothetical ancestral genotype, the extended
hif
cluster. The
hif
region of INT1, an invasive nontypeable isolate, resembles the hypothetical ancestor. We propose that a progenitor strain acquired the extended cluster by horizontal transfer and that other variants arose as deletions. The structure of the
hif
cluster may correlate with colonization site or pathogenicity.
Effect of Temperature on In Vivo Protein Synthetic Capacity in
Escherichia coli
Authors: Farewell Anne , Neidhardt Frederick C. 
Published: December 2019

ABSTRACT

In this report, we examine the effect of temperature on protein synthesis. The rate of protein accumulation is determined by three factors: the number of working ribosomes, the rate at which ribosomes are working, and the rate of protein degradation. Measurements of RNA/protein ratios and the levels of individual ribosomal proteins and rRNA show that the cellular amount of ribosomal machinery in
Escherichia coli
is constant between 25 and 37°C. Within this range, in a given medium, temperature affects ribosomal function the same as it affects overall growth. Two independent methodologies show that the peptide chain elongation rate increases as a function of temperature identically to growth rate up to 37°C. Unlike the growth rate, however, the elongation rate continues to increase up to 44°C at the same rate as between 25 and 37°C. Our results show that the peptide elongation rate is not rate limiting for growth at high temperature. Taking into consideration the number of ribosomes per unit of cell mass, there is an apparent excess of protein synthetic capacity in these cells, indicating a dramatic increase in protein degradation at high temperature. Temperature shift experiments show that peptide chain elongation rate increases immediately, which supports a mechanism of heat shock response induction in which an increase in unfolded, newly translated protein induces this response. In addition, we find that at low temperature (15°C), cells contain a pool of nontranslating ribosomes which do not contribute to cell growth, supporting the idea that there is a defect in initiation at low temperature.
Topological Model of the
Rhodobacter capsulatus
Light-Harvesting Complex I Assembly Protein LhaA (Previously Known as ORF1696)
Authors: Young C. S. , Beatty J. T. 
Published: December 2019

ABSTRACT

A theoretical topology of the
Rhodobacter capsulatus
membrane protein LhaA was formulated and evaluated by gene fusion experiments. The apparent topological locations of fusion enzymes were compared with the theoretically derived structure, and a model of LhaA is suggested that consists of 12 transmembrane segments, with the N and C termini residing in the cytoplasm.
Transcription Analysis of Two Disparate rRNA Operons in the Halophilic ArchaeonHaloarcula marismortui
Authors: Dennis Patrick P. , Ziesche Sonia , Mylvaganam Shanthini 
Published: December 2019

ABSTRACTThe genome of the halophilic archaeonHaloarcula marismortuicontains two rRNA operons designatedrrnAandrrnB. Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized. The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated. The two operons exhibit other profound differences that include (i) the presence inrrnAand the absence inrrnBof tRNAAlaand tRNACysgenes in the intergenic and distal regions, respectively, (ii) divergent 5′ flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA fromrrnAoperon transcripts and of 23S rRNA fromrrnBoperon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA fromrrnBoperon transcripts follows an unusual and different pathway that is apparently devoid of any 5′ processing intermediate.
Characterization of a Novel Acyl Carrier Protein, RkpF, Encoded by an Operon Involved in Capsular Polysaccharide Biosynthesis in
Sinorhizobium meliloti
Authors: Epple Guido , van der Drift Koen M. G. M. , Thomas-Oates Jane E. , Geiger Otto 
Published: December 2019

ABSTRACT

Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in
Sinorhizobium meliloti
AK631 functional
rkpABCDEF
genes are required for the production of RKPs. After cloning the
rkpF
gene, we overexpressed and purified the derived protein product (RkpF) in
Escherichia coli
. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive β-alanine added to the growth medium. If homogeneous RkpF protein is incubated with radiolabeled coenzyme A in the presence of purified holo-ACP synthase from
E. coli
, an in vitro transfer of 4′-phosphopantetheine to the RkpF protein can be observed. The conversion from apo-RkpF protein to holo-RkpF protein seems to go along with a major conformational change of the protein structure, because the holo-RkpF protein runs significantly faster on native polyacrylamide gel electrophoresis than the apo-RkpF protein. Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF protein and a mass of 9,927 for the holo-RkpF protein. Our data show that RkpF is a novel ACP.
IS
481
and IS
1002
of
Bordetella pertussis
Create a 6-Base-Pair Duplication upon Insertion at a Consensus Target Site
Authors: Stibitz Scott 
Published: December 2019

ABSTRACT

The insertion sequence IS
481
and its isoform IS
1002
have been observed to transpose into the
bvgAS
locus of
Bordetella pertussis
, for which the DNA sequence has previously been determined. Upon insertion of IS
481
at three different sites and IS
1002
at one site, a 6-bp sequence originally present was found at the junction of
bvg
and insertion sequence DNA. This indicates that, contrary to prior reports, IS
481
and IS
1002
do create a duplication upon insertion. In this light, examination of these and other examples of IS
481
and IS
1002
reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS
481
or IS
1002
, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.
Limonene-1,2-Epoxide Hydrolase from
Rhodococcus erythropolis
DCL14 Belongs to a Novel Class of Epoxide Hydrolases
Authors: van der Werf Mariët J. , Overkamp Karin M. , de Bont Jan A. M. 
Published: December 2019

ABSTRACT

An epoxide hydrolase from
Rhodococcus erythropolis
DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when
R. erythropolis
is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50°C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4′-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1,10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.
Mutation Analysis of PobR and PcaU, Closely Related Transcriptional Activators in
Acinetobacter
Authors: Kok Ruben G. , D’Argenio David A. , Ornston L. Nicholas 
Published: December 2019

ABSTRACT

Acinetobacter
PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite
p
-hydroxybenzoate and activates transcription of
pobA
, the structural gene for the enzyme that converts
p
-hydroxybenzoate to protocatechuate. This compound, differing from
p
-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from
Acinetobacter
strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which
pobR
function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the
pobA-pobR
intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on
p
-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.