bluuebunny/crossref_metadata_2025 huggingface dataset:
The
d
-Xylose-Binding Protein, XylF, from
Thermoanaerobacter ethanolicus
39E: Cloning, Molecular Analysis, and Expression of the Structural Gene
Authors: Erbeznik Milutin , Strobel Herbert J. , Dawson Karl A. , Jones Chris R.
Published: December 2019

Immediately downstream from the
Thermoanaerobacter ethanolicus xylAB
operon, comprising genes that encode
d
-xylose isomerase and
d
-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the
Escherichia coli
d
-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent
d
-xylose transport. The 25-residue N-terminal fragment of the deduced
T. ethanolicus
XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [
14
C]palmitate. The induced
E. coli
avidly bound
d
-[
14
C]xylose, yielding additional evidence that
T. ethanolicus
XylF is the
d
-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)
3
IX
3
(DN)(SGP)X
3
(GS)X(LIVA)
2
X
2
A. Transcription of the monocistronic 1.3-kb
xylF
mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that
xylF
transcription initiates from two +1 sites, both situated within the
xylAB
operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity
d
-xylose uptake system are not located in the vicinity of
xylF
in
T. ethanolicus
. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.
A Region in the
Bacillus subtilis
Transcription Factor Spo0A That Is Important for
spoIIG
Promoter Activation
Authors: Buckner Cindy M. , Schyns Ghislain , Moran Charles P.
Published: December 2019

Spo0A is a DNA binding protein in
Bacillus subtilis
required for the activation of
spoIIG
and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ς
A
subunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ς
A
, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ς
A
. We also found that the S231F substitution partially restored use of the
spoIIG
promoter by the ς
A
H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for
spoIIG
promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of
abrB
transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the
spoIIA
promoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ς
H
-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ς
A
-dependent promoter activation.
Molecular Characterization and Postsplicing Fate of Three Introns within the Single rRNA Operon of the Hyperthermophilic Archaeon
Aeropyrum pernix
K1
Authors: Nomura Norimichi , Sako Yoshihiko , Uchida Aritsune
Published: December 2019

The single rRNA operon (
arnS-arnL
) of the hyperthermophilic archaeon
Aeropyrum pernix
K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Iα was located at position 908 (
Escherichia coli
numbering [H. F. Noller, Annu. Rev. Biochem. 53:119–162, 1984]) of the 16S rRNA, while the 202-nt intron Iβ and 575-nt intron Iγ were located at positions 1085 and 1927 (
E. coli
numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in
E. coli
. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Iα-, Iβ-, and Iγ-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Iα and Iγ, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.
Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium
Azoarcus anaerobius
Authors: Philipp Bodo , Schink Bernhard
Published: December 2019

The denitrifying bacterium
Azoarcus anaerobius
LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K
3
Fe(CN)
6
as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein
−1
for resorcinol hydroxylation and 150 mU mg of protein
−1
for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.
General Stress Transcription Factor ς
B
and Its Role in Acid Tolerance and Virulence of
Listeria monocytogenes
Authors: Wiedmann Martin , Arvik Torey J. , Hurley Richard J. , Boor Kathryn J.
Published: December 2019

The gene encoding the general stress transcription factor ς
B
in the gram-positive bacterium
Listeria monocytogenes
was isolated with degenerate PCR primers followed by inverse PCR amplification. Evidence for gene identification includes the following: (i) phylogenetic analyses of reported amino acid sequences for ς
B
and the closely related ς
F
proteins grouped
L. monocytogenes
ς
B
in the same cluster with the ς
B
proteins from
Bacillus subtilis
and
Staphylococcus aureus
, (ii) the gene order in the 2,668-bp portion of the
L. monocytogenes sigB
operon is
rsbU-rsbV-rsbW-sigB-rsbX
and is therefore identical to the order of the last five genes of the
B. subtilis sigB
operon, and (iii) an
L. monocytogenes
ς
B
mutant had reduced resistance to acid stress in comparison with its isogenic parent strain. The
sigB
mutant was further characterized in mouse models of listeriosis by determining recovery rates of the wild-type and mutant strains from livers and spleens following intragastric or intraperitoneal infection. Our results suggest that ς
B
-directed genes do not appear to be essential for the spread of
L. monocytogenes
to mouse liver or spleen at 2 and 4 days following intragastric or intraperitoneal infection.
ftsE
(Ts) Affects Translocation of K
+
-Pump Proteins into the Cytoplasmic Membrane of
Escherichia coli
Authors: Ukai Hideki , Matsuzawa Hiroshi , Ito Koreaki , Yamada Mamoru , Nishimura Akiko
Published: December 2019

The
ftsE
(Ts) mutation of
Escherichia coli
causes defects in cell division and cell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of KdpA, Kup, and TrkH, all of which proved functional in vivo as K
+
ion pumps, in the mutant cells. During growth at 41°C, these proteins were progressively lost from the membrane fraction. The reduction in the abundance of these proteins inversely correlated with cell growth, but the preformed proteins in the membrane were stable at 41°C, indicating that the molecules synthesized at the permissive temperature were diluted in a growth-dependent manner at a high temperature. Pulse-chase experiments showed that KdpA-PhoA was synthesized, but the synthesized protein did not translocate into the membrane of the ftsE(Ts) cells at 41°C and degraded very rapidly. The loss of KdpA-PhoA from the membrane fractions of ftsE(Ts) cells was suppressed by a multicopy plasmid carrying the
ftsE
+
gene. While cell growth stopped when the abundance of these proteins decreased 15-fold, the addition of a high concentration of K
+
ions specifically alleviated the growth defect of ftsE(Ts) cells but not cell division, and the cells elongated more than 100-fold. We conclude that one of the causes of growth cessation in the ftsE(Ts) mutants is a defect in the translocation of K
+
-pump proteins into the cytoplasmic membrane.
The Regulated Outer Membrane Protein Omp21 from
Comamonas acidovorans
Is Identified as a Member of a New Family of Eight-Stranded β-Sheet Proteins by Its Sequence and Properties
Authors: Baldermann C. , Lupas A. , Lubieniecki J. , Engelhardt H.
Published: December 2019

Omp21, a minor outer membrane protein of the soil bacterium
Comamonas acidovorans
, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene
omp21
encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of β structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic β strands, which includes virulence proteins, such as the neisserial opacity proteins,
Salmonella typhimurium
Rck, and
Yersinia enterocolitica
Ail, as well as the major outer membrane proteins OmpA from
Escherichia coli
and OprF from
Pseudomonas aeruginosa
.
Escherichia coli
Strains Lacking Protein HU Are UV Sensitive due to a Role for HU in Homologous Recombination
Authors: Li Shisheng , Waters Raymond
Published: December 2019

hupA
and
hupB
encode the α and β subunits of the
Escherichia coli
histone-like protein HU. Here we show that
E. coli hup
mutants are sensitive to UV in the

rec
+
sbc

+
,
recBC sbcA
,
recBC sbcBC
,
umuDC
,
recF
, and
recD
backgrounds. However,
hupAB
mutations do not enhance the UV sensitivity of resolvase-deficient
recG ruvA
strains.
hupAB uvrA
and
hupAB recG
strains are supersensitive to UV.
hup
mutations enhance the UV sensitivity of
ruvA
strains to a much lesser extent but enhance that of
rus-1 ruvA
strains to the same extent as for

rus
+
ruv

+
strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity of
hup
mutants cannot be suppressed by overexpression of wild-type or mutant
gyrB
, which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.
Lack of Consistent Short Sequence Repeat Polymorphisms in Genetically Homologous Colonizing and Invasive
Candida albicans
Strains
Authors: Lunel Frans Verduyn , Licciardello Lidia , Stefani Stefania , Verbrugh Henri A. , Melchers Willem J. G. , Meis Jacques F. G. M. , Scherer Stewart , van Belkum Alex
Published: December 2019

Short sequence repeats (SSRs), potentially representing variable numbers of tandem repeat (VNTR) loci, were identified for the human-pathogenic yeast species
Candida albicans
by computerized DNA sequence scanning. The individual SSR regions were investigated in different clinical isolates of
C. albicans
. Most of the
C. albicans
SSRs were identified as genuine VNTRs. They appeared to be present in multiple allelic variants and were demonstrated to be diverse in length among nonrelated strains. As such, these loci provide adequate targets for the molecular typing of
C. albicans
strains. VNTRs encountered in other microbial species sometimes participate in regulation of gene expression and function as molecular switches at the transcriptional or translational level. Interestingly, the VNTRs identified here often encode polyglutamine stretches and are frequently located within genes potentially involved in the regulation of transcription. DNA sequencing of these VNTRs demonstrated that the length variability was restricted to the CAA/CAG repeats encoding the polyglutamine stretches. For these reasons, paired
C. albicans
isolates of similar genotype, either found as noninvasive colonizers or encountered in an invasive state in the same individual, were studied with respect to potentially invasion-related alterations in the VNTR profiles. However, none of the VNTRs analyzed thus far varied systematically with the transition from colonization to invasion. In contrast to the situation described for some prokaryotic species, this finding suggests that VNTRs of
C. albicans
may not simply function as contingency loci related to straightforward on/off regulation of invasion-related gene expression.
Disruption of the
Candida albicans TPS1
Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity
Authors: Zaragoza Oscar , Blazquez Miguel A. , Gancedo Carlos
Published: December 2019

The
TPS1
gene from
Candida albicans
, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a
tps1
mutant from
Saccharomyces cerevisiae
. In contrast with the wild-type strain, the double
tps1/tps1
disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the
tps1/tps1
disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10
6
CFU of the
tps1/tps1
mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.
Physical and Genetic Map of the Obligate Intracellular Bacterium
Coxiella burnetii
Authors: Willems H. , Jäger Cornelie , Baljer Georg
Published: December 2019

Pulsed-field gel electrophoresis and PCR techniques have been used to construct a
Not
I macrorestriction map of the obligate intracellular bacterium
Coxiella burnetii
Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29
Not
I restriction fragments. The average resolution is 72.5 kb, or about 3.5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative
oriC
has been located on a 27.5-kb
Not
I fragment. Gene organization upstream the
oriC
is almost identical to that of
Pseudomonas putida
and
Bacillus subtilis
, whereas gene organization downstream the
oriC
seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.
Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1
Authors: Payne Jason W. , Bolton Harvey , Campbell James A. , Xun Luying
Published: December 2019

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O
2
were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH
2
and O
2
. The FMN reductase provided EDTA monooxygenase with FMNH
2
by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C.

K
m

s were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA
2−
; this difference in

K
m

indicates that the enzyme has greater affinity for MgEDTA
2−
. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH
2
-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.
Regulation of Porin-Mediated Outer Membrane Permeability by Nutrient Limitation in
Escherichia coli
Authors: Liu XueQiao , Ferenci Thomas
Published: December 2019

OmpF and OmpC porins were differentially regulated by nutrient limitation and growth rate in glucose- or nitrogen-limited chemostat cultures of
Escherichia coli
. Transcriptional and translational
ompF
fusions showed a sharp peak of expression under glucose limitation at
D
= 0.3 h
−1
, with lower amounts at lower and higher growth rates. The peak of OmpR-dependent transcriptional stimulation of
ompF
under glucose limitation in minimal salts media was about 20-fold above nutrient excess levels and 3-fold higher than that achieved with low osmolarity. Analysis of outer membrane protein levels and results of growth competition experiments with porin mutants were consistent with the enhanced role of OmpF under glucose limitation, but not N limitation. In contrast, OmpC was the major porin under N limitation but was increasingly expressed under glucose limitation at very low growth rates approaching starvation, when OmpF was downregulated. In summary, outer membrane permeability under N-limited, sugar-rich conditions is largely based on OmpC, whereas porin activity is a complex, highly sensitive function of OmpF, OmpC, and LamB glycoporin expression under different levels of glucose limitation. Indeed, the OmpF level was more responsive to nutrient limitation than to medium osmolarity and suggested a significant additional layer of control over the porin-regulatory network.
Effect of Oxygen on Translation and Posttranslational Steps in Expression of Photosynthesis Genes in
Rhodobacter capsulatus
Authors: Hebermehl Markus , Klug Gabriele
Published: December 2019

The formation of the photosynthetic apparatus in
Rhodobacter capsulatus
is regulated by oxygen tension. Previous studies have shown a regulatory effect of oxygen on the transcription of photosynthesis genes and on the stability of certain mRNA segments. Here we show that oxygen affects
puf
and
puc
gene expression posttranslationally and that this regulation depends on the presence of bacteriochlorophyll. Our data suggest that this posttranslational effect of oxygen on
puf
and
puc
expression is due to the primary effect of oxygen on bacteriochlorophyll synthesis or assembly of pigment protein complexes. Oxygen does not affect the rates of translation of
puf
-encoded proteins.
Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding
Authors: Dantley Kathi A. , Dannelly H. Kathleen , Burdett Vickers
Published: December 2019

Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.
Rhs
Elements Comprise Three Subfamilies Which Diverged Prior to Acquisition by
Escherichia coli
Authors: Wang Yong-Dong , Zhao Sheng , Hill Charles W.
Published: December 2019

The
Rhs
elements are complex genetic composites widely spread among
Escherichia coli
isolates. One of their components, a 3.7-kb, GC-rich core, maintains a single open reading frame that extends the full length of the core and then 400 to 600 bp beyond into an AT-rich region. Whereas
Rhs
cores are homologous, core extensions from different elements are dissimilar. Two new
Rhs
elements from strains of the ECOR reference collection have been characterized.
RhsG
(from strain ECOR-11) maps to min 5.3, and
RhsH
(from strain ECOR-45) maps to min 32.8, where it lies in tandem with
RhsE
. Comparison of strain K-12 to ECOR-11 indicates that
RhsG
was once present in but has been largely deleted from an ancestor of K-12. Phylogenetic analysis shows that the cores from eight known elements fall into three subfamilies,
RhsA-B-C-F
,
RhsD-E
, and
RhsG-H
. Cores from different subfamilies diverge 22 to 29%. Analysis of substitutions that distinguish between subfamilies shows that the origin of the ancestral core as well as the process of subfamily separation occurred in a GC-rich background. Furthermore, each subfamily independently passed from the GC-rich background to a less GC-rich background such as
E. coli
. A new example of core-extension shuffling provides the first example of exchange between cores of different subfamilies. A novel component of
RhsE
and
RhsG
,
vgr
, encodes a large protein distinguished by 18 to 19 repetitions of a Val-Gly dipeptide occurring with a eight-residue periodicity.
TherelA/spoT-Homologous Gene inStreptomyces coelicolorEncodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities
Authors: Martínez-Costa Oscar H. , Fernández-Moreno Miguel A. , Malpartida Francisco
Published: December 2019

Streptomyces coelicolor(p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of therelgene have been examined.S. coelicolorRel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-definedEscherichia coli relAandrelA/spoTmutants provides evidence thatS. coelicolorRel, like nativeE. coliRelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolorC-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as theE. colitruncated RelA protein (1 to 455 amino acids). AnE. coli relA spoTdouble deletion mutant transformed withS. coelicolor relgene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay inE. coliexpressing theS. coelicolor relgene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains ofS. coelicolorRel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relAin anS. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerelgene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.
Influence of the Secondary Cell Wall Polymer on the Reassembly, Recrystallization, and Stability Properties of the S-Layer Protein from
Bacillus stearothermophilus
PV72/p2
Authors: Sára Margit , Dekitsch Christine , Mayer Harald F. , Egelseer Eva M. , Sleytr Uwe B.
Published: December 2019

The high-molecular-weight secondary cell wall polymer (SCWP) from
Bacillus stearothermophilus
PV72/p2 is mainly composed of
N
-acetylglucosamine (GlcNAc) and
N
-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 μg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-
l
-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.
The
Escherichia coli
Citrate Carrier CitT: a Member of a Novel Eubacterial Transporter Family Related to the 2-Oxoglutarate/Malate Translocator from Spinach Chloroplasts
Authors: Pos Klaas Martinus , Dimroth Peter , Bott Michael
Published: December 2019

Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol,
Escherichia coli
converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated
citT
) located at 13.90 min on the
E. coli
chromosome between
rna
and the citrate lyase genes encodes a citrate carrier.
E. coli
transformed with a plasmid expressing
citT
was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in
E. coli
, it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the
E. coli
CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.
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

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

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

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

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

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

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

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

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

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

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

The 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

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.