bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pseudomonas aeruginosa fur
Overlaps with a Gene Encoding a Novel Outer Membrane Lipoprotein, OmlA
Authors: Ochsner Urs A. , Vasil Adriana I. , Johnson Zaiga , Vasil Michael L. 
Published: December 2019

ABSTRACT

A novel outer membrane lipoprotein in
Pseudomonas aeruginosa
is encoded by the
omlA
gene, which was identified immediately upstream of the
fur
(ferric uptake regulator) gene. The
omlA
and
fur
genes were divergently transcribed and had overlapping promoter regions. The proximal
fur
P2 promoter and the
omlA
promoter shared a 5-bp DNA motif for their −10 promoter elements. The distal
fur
P1 promoter was located within the
omlA
coding sequence, and the
omlA
and
fur
T1 mRNAs overlapped by 154 nucleotides. Optimal expression of both
fur
and
omlA
required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence of
cis
-acting transcriptional activation elements located within the
omlA
and
fur
genes, respectively. The levels of Fur and OmlA proteins had no influence on
omlA
or
fur
expression, excluding any
trans
-acting cross-regulation between
fur
and
omlA
. Expression of
omlA
was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4.5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of
Escherichia coli
,
Vibrio cholerae
, and
Haemophilus influenzae
, a protein of unknown function. All
P. aeruginosa
strains tested as well as
Pseudomonas fluorescens
were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping
fur
gene was constructed. The
omlA
mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed.
Cell Cycle Control of a Holdfast Attachment Gene in
Caulobacter crescentus
Authors: Janakiraman Raji S. , Brun Yves V. 
Published: December 2019

ABSTRACT

Attachment to surfaces by the prosthecate bacterium
Caulobacter crescentus
is mediated by an adhesive organelle, the holdfast, found at the tip of the stalk. Indirect evidence suggested that the holdfast first appears at the swarmer pole of the predivisional cell. We used fluorescently labeled lectin and transmission electron microscopy to detect the holdfast in different cell types. While the holdfast was readily detectable in stalked cells and at the stalked poles of predivisional cells, we were unable to detect the holdfast in swarmer cells or at the flagellated poles of predivisional cells. This suggests that exposure of the holdfast to the outside of the cell occurs during the differentiation of swarmer to stalked cells. To investigate the timing of holdfast synthesis and exposure to the outside of the cell, we have examined the regulation of a holdfast attachment gene,
hfaA
. The
hfaA
gene is part of a cluster of four genes (
hfaABDC
), identified in strain CB2A and involved in attachment of the holdfast to the polar region of the cell. We have identified the
hfaA
gene in the synchronizable
C. crescentus
strain CB15. The sequence of the CB2A
hfaA
promoter suggested that it was regulated by ς
54
. We show that the transcription of
hfaA
from either strain is not dependent on ς
54
. Using a
hfaA-lacZ
fusion, we show that the transcription of
hfaA
is temporally regulated during the cell cycle, with maximal expression in late-predivisional cells. This increase in expression is largely due to the preferential transcription of
hfaA
in the swarmer pole of the predivisional cell.
Spectra of Spontaneous Growth-Dependent and Adaptive Mutations at
ebgR
Authors: Hall Barry G. 
Published: December 2019

ABSTRACT

A comparison of the spectra of spontaneous growth-dependent and adaptive mutations in
ebgR
shows that both spectra are dominated by insertion sequence (IS)-mediated mutations. The difference between growth-dependent mutations (61% IS mediated) and adaptive mutations (80% IS mediated) is highly significant (
P
< 0.0001). In contrast, the spectra of growth-dependent and adaptive non-IS-mediated mutations do not differ from each other and therefore do not provide support for the hypothesis that adaptive and growth-dependent mutations arise by substantially different mechanisms.
Gene Targeting inPenicillium chrysogenum: Disruption of thelys2Gene Leads to Penicillin Overproduction
Authors: Casqueiro Javier , Gutiérrez Santiago , Bañuelos Oscar , Hijarrubia Maria Jose , Martín Juan Francisco 
Published: December 2019

ABSTRACTTwo strategies have been used for targeted integration at thelys2locus ofPenicillium chrysogenum. In the first strategy the disruption oflys2was obtained by a single crossing over between the endogenouslys2and a fragment of the same gene located in an integrative plasmid.lys2-disrupted mutants were obtained with 1.6% efficiency when thelys2homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly,lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using twolys2homologous regions of 4.3 and 3.0 kb flanking thepyrGmarker. No homologous recombination was observed when the selectable marker was flanked by shortlys2homologous DNA fragments. The disruption oflys2was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. Thelys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded bylys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.
Two Nucleotide Transport Proteins in
Chlamydia trachomatis
, One for Net Nucleoside Triphosphate Uptake and the Other for Transport of Energy
Authors: Tjaden J. , Winkler H. H. , Schwöppe C. , Van Der Laan M. , Möhlmann T. , Neuhaus H. E. 
Published: December 2019

ABSTRACT

The genome of
Chlamydia trachomatis
, one of the most prominent human pathogens, contains two structural genes coding for proteins, herein called Npt1
Ct
and Npt2
Ct
(nucleoside phosphate transporters 1 and 2 of
C. trachomatis
), exhibiting 68 and 61% similarity, respectively, to the ATP/ADP transporter from the intracellular bacterium
Rickettsia prowazekii
at the deduced amino acid level. Hydropathy analysis and sequence alignments suggested that both proteins have 12 transmembrane domains. The putative transporters were expressed as histidine-tagged proteins in
Escherichia coli
to study their biochemical properties. His
10
-Npt1
Ct
catalyzed ATP and ADP transport in an exchange mode. The apparent K
m
values were 48 (ATP) and 39 (ADP) μM. ATP and ADP transport was specific since AMP, GTP, CTP, UTP, dATP, dCTP, dGTP, and dTTP did not inhibit uptake. In contrast, His
10
-Npt2
Ct
transported all four ribonucleoside triphosphates with apparent K
m
values of 31 μM (GTP), 302 μM (UTP), 528 μM (CTP), and 1,158 μM (ATP). Ribonucleoside di- and monophosphates and deoxyribonucleotides were not substrates. The protonophore m -chlorocarbonylcyanide phenylhydrazone abolished uptake of all nucleoside triphosphates by Npt2
Ct
. This observation indicated that His
10
-Npt2
Ct
acts as a nucleosidetriphosphate/H
+
symporter energized by the proton motive force across the
Escherichia coli
cytoplasmic membrane. We conclude that Npt1
Ct
provides chlamydiae with energy whereas Npt2
Ct
catalyzes the net uptake of ribonucleoside triphosphates required for anabolic reactions.
Specific Effects of a
recB
Mutation on the HfrH Strain of
Escherichia coli
Authors: Đermić Damir , Trgovčević Željko 
Published: December 2019

ABSTRACT

The
recB268
::Tn
10
mutation was introduced into the HfrH strain of
Escherichia coli
. Compared with
recB
F
−
and
recB
F
+
cells, the viability of this mutant strain was much lower. Compared with wild-type HfrH, the
recB
derivative donated much shorter fragments of its chromosome to the recipient. It is suggested that the
recB
gene product (i.e., RecBCD enzyme) participates in Hfr transfer.
Nitrate-Dependent Regulation of Acetate Biosynthesis and Nitrate Respiration by
Clostridium thermoaceticum
Authors: Arendsen Alexander F. , Soliman Mohsin Q. , Ragsdale Stephen W. 
Published: December 2019

ABSTRACT

Nitrate has been shown to shunt the electron flow in
Clostridium thermoaceticum
from CO
2
to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J. M. Fröstl, C. Seifritz, and H. L. Drake, J. Bacteriol. 178:4597–4603, 1996). Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway. The CO oxidation activity in crude extracts of nitrate (30 mM)–supplemented cultures was fivefold less than that of nitrate-free cultures, while the H
2
oxidation activity was six- to sevenfold lower. The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis. Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures. Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%). Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged. Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level. At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered. We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min
−1
mg
−1
) in crude extracts of
C. thermoaceticum
. Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins. CO
2
also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity.
Secretion, Localization, and Antibacterial Activity of TasA, a
Bacillus subtilis
Spore-Associated Protein
Authors: Stöver Axel G. , Driks Adam 
Published: December 2019

ABSTRACT

The synthesis and subcellular localization of the proteins that comprise the
Bacillus subtilis
spore are under a variety of complex controls. To better understand these controls, we have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore. TasA synthesis begins approximately 30 min after the onset of sporulation and requires the sporulation transcription factor genes
spo0H
and
spo0A
. The first 81 nucleotides of
tasA
encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from TasA isolated from a sporulating cell lysate. In
B. subtilis
cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores. Cells unable to produce SipW produce a 34-kDa form of TasA, consistent with a failure to remove the N-terminal 27 amino acids. In cells engineered to express
sipW
and
tasA
during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium. These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores. Although TasA is dispensable for sporulation under laboratory conditions, we find that TasA has a broad-spectrum antibacterial activity. We discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus conferring a competitive advantage to the spore.
Aspartate Kinase-Independent Lysine Synthesis in an Extremely Thermophilic Bacterium,
Thermus thermophilus
: Lysine Is Synthesized via α-Aminoadipic Acid Not via Diaminopimelic Acid
Authors: Kobashi Nobuyuki , Nishiyama Makoto , Tanokura Masaru 
Published: December 2019

ABSTRACT

An aspartate kinase-deficient mutant of
Thermus thermophilus
, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that
T. thermophilus
contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in
T. thermophilus
, lysine auxotrophic mutants of
T. thermophilus
were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of α-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A
Bam
HI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed
hacA
and
hacB
, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of
hacA
yielded mutants showing lysine auxotrophy which was restored by addition of α-aminoadipic acid or α-ketoadipic acid. All of these results indicated that in
T. thermophilus
, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using α-aminoadipic acid as a biosynthetic intermediate.
Sequence of Shiga Toxin 2 Phage 933W fromEscherichia coliO157:H7: Shiga Toxin as a Phage Late-Gene Product
Authors: Plunkett Guy , Rose Debra J. , Durfee Timothy J. , Blattner Frederick R. 
Published: December 2019

ABSTRACTLysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogenEscherichia coliO157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene,stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein ofYersiniaspecies, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology tolom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, andbor, implicated in serum resistance.
Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein inClostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp
Authors: Pagès Sandrine , Bélaïch Anne , Fierobe Henri-Pierre , Tardif Chantal , Gaudin Christian , Bélaïch Jean-Pierre 
Published: December 2019

ABSTRACTThe gene encoding the scaffolding protein of the cellulosome fromClostridium cellulolyticum, whose partial sequence was published earlier (S. Pagès, A. Bélaı̈ch, C. Tardif, C. Reverbel-Leroy, C. Gaudin, and J.-P. Bélaı̈ch, J. Bacteriol. 178:2279–2286, 1996; C. Reverbel-Leroy, A. Bélaı̈ch, A. Bernadac, C. Gaudin, J. P. Bélaı̈ch, and C. Tardif, Microbiology 142:1013–1023, 1996), was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. In addition, a second gene located 8.89 kb downstream ofcipCwas found to encode a three-domain protein, called ORFXp, which includes a cohesin domain. By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane ofC. cellulolyticumand is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka= 4.8 × 109M−1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lowerKafor CelA (2.6 × 108M−1). Taken together, these data suggest that ORFXp may play a role in cellulosome assembly.
Purification and Characterization of Two Extremely Thermostable Enzymes, Phosphate Acetyltransferase and Acetate Kinase, from the Hyperthermophilic Eubacterium
Thermotoga maritima
Authors: Bock Anne-Katrin , Glasemacher Jürgen , Schmidt Roland , Schönheit Peter 
Published: December 2019

ABSTRACT

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium
Thermotoga maritima
have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (α
4
) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from
Clostridium acetobutylicum
rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + P
i
⇌ acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent

K
m

values for acetyl-CoA, P
i
, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 μM, respectively; the apparent
V
max
values (at 55°C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90°C and was not inactivated by heat upon incubation at 80°C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (α
2
) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon
Methanosarcina thermophila
. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP ⇌ acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent

K
m

values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent
V
max
values (at 50°C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn
2+
and Mg
2+
being most effective. The enzyme had a temperature optimum at 90°C and was stabilized against heat inactivation by salts. In the presence of (NH
4
)
2
SO
4
(1 M), which was most effective, the enzyme did not lose activity upon incubation at 100°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.
The Cyclic AMP Receptor Protein Is Dependent on GcvA for Regulation of the
gcv
Operon
Authors: Wonderling Laura D. , Stauffer George V. 
Published: December 2019

ABSTRACT

The
Escherichia coli gcv
operon is transcriptionally regulated by the GcvA, GcvR, Lrp, and PurR proteins. In this study, the cyclic AMP (cAMP) receptor protein (CRP) is shown to be involved in positive regulation of the
gcv
operon. A
crp
deletion reduced expression of a
gcvT-lacZ
fusion almost fourfold in glucose minimal (GM) medium. The phenotype was complemented by both the wild-type
crp
gene and four
crp
alleles that encode proteins with amino acid substitutions in known activating regions of CRP. A
cyaA
deletion also resulted in a fourfold decrease in
gcvT-lacZ
expression, and wild-type expression was restored by the addition of cAMP to the growth medium. A
cyaA crp
double deletion resulted in levels of
gcvT-lacZ
expression identical to those observed with either single mutation, showing that CRP and cAMP regulate through the same mechanism. Growth in GM medium plus cAMP or glycerol minimal medium did not result in a significant increase in
gcvT-lacZ
expression. Thus, the level of cAMP present in GM medium appears to be sufficient for regulation by CRP. DNase I footprint analysis showed that CRP binds and protects two sites centered at bp −313 (site 1) and bp −140 (site 2) relative to the transcription initiation site, but a mutational analysis demonstrated that only site 1 is required for CRP-mediated regulation of
gcvT-lacZ
expression. Expression of the
gcvT-lacZ
fusion in a
crp gcvA
double mutant suggested that CRP’s role is dependent on the GcvA protein.
Identification and Cloning of an
Erwinia carotovora
subsp.
carotovora
Bacteriocin Regulator Gene by Insertional Mutagenesis
Authors: Chuang Duen-Yau , Kyeremeh Ampaabeng G. , Gunji Yuichi , Takahara Yoshiyuki , Ehara Yoshio , Kikumoto Toshio 
Published: December 2019

ABSTRACT

Avirulent
Erwinia carotovora
subsp.
carotovora
CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn
5
insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn
5
insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3′ end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5′ end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with
Yersinia enterocolitica
Yrp, a regulator of enterotoxin (Y-ST) production;
Escherichia coli
host factor 1, required for Q
β
-replicase; and
Azorhizobium caulinodans
NrfA, required for the expression of
nifA
. ORF2 was designated
brg
, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into
E. coli
DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.