bluuebunny/crossref_metadata_2025 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

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

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

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

Two 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

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

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

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

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

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

Lysogenic 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

The 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

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

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

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.
The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in
Rhodobacter capsulatus
Is Correlated with Cellular Nitrogen Status
Authors: Yakunin Alexander F. , Laurinavichene Tatyana V. , Tsygankov Anatoly A. , Hallenbeck Patrick C.
Published: December 2019

The photosynthetic bacterium
Rhodobacter capsulatus
has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH
4
+
. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH
4
+
, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH
4
+
, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N
2
or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in
R. capsulatus
the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.
Purification of a Glutathione
S
-Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Kingma Jaap , Kruizinga Wim , Janssen Dick B.
Published: December 2019

A glutathione
S
-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) and
cis
-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium
Rhodococcus
sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and
cis
-1,2-dichloroepoxyethane, with
V
max
values of 66 and 2.4 μmol min
−1
mg of protein
−1
and

K
m

values of 0.3 and 0.1 mM for isoprene monoxide and
cis
-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM.
1
H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD
+
-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD
+
and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.
Oxygen Depletion-Induced Dormancy in
Mycobacterium bovis
BCG
Authors: Lim Amanda , Eleuterio Marianne , Hutter Bernd , Murugasu-Oei Bernadette , Dick Thomas
Published: December 2019

Gradual depletion of oxygen causes the shift-down of aerobic growing
Mycobacterium bovis
BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and α-crystallin-like protein and is sensitive to metronidazole.
Characterization of
mdcR
, a Regulatory Gene of the Malonate Catabolic System in
Klebsiella pneumoniae
Authors: Peng Hwei-Ling , Shiou Sheng-Ru , Chang Hwan-You
Published: December 2019

The
Klebsiella pneumoniae mdcR
gene, which encodes a LysR-type regulator, was overexpressed in
Escherichia coli
. Purified MdcR was found to bind specifically to the control region of either the malonate decarboxylase (
mdc
) genes or
mdcR
. We have also demonstrated that MdcR is an activator of the expression of the
mdc
genes, whereas it represses the transcription of the putative control region of
mdcR
, P

mdcR

, indicating a negative autoregulatory control.
Genetic Diversity in the Protective Antigen Gene of
Bacillus anthracis
Authors: Price Lance B. , Hugh-Jones Martin , Jackson Paul J. , Keim Paul
Published: December 2019

Bacillus anthracis
is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (
pag
) was sequenced from 26 of the most diverse
B. anthracis
strains to identify potential variation in the toxin and to further our understanding of
B. anthracis
evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of
pag
from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the
pag
corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in
B. anthracis
has occurred with little or no horizontal transfer between the different strains.
Expression of a Germination-Specific Amidase, SleB, of Bacilli in the Forespore Compartment of Sporulating Cells and Its Localization on the Exterior Side of the Cortex in Dormant Spores
Authors: Moriyama Ryuichi , Fukuoka Hideyuki , Miyata Shigeru , Kudoh Sachio , Hattori Atsuhiko , Kozuka Satoshi , Yasuda Yoko , Tochikubo Kunio , Makino Shio
Published: December 2019

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The
sleB
gene encoding this amidase in
Bacillus subtilis
and
Bacillus cereus
was expressed in the forespore compartment of sporulating cells under the control of ς
G
, as shown by Northern blot and primer extension analyses. The forespore-specific expression of
B. subtilis sleB
was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both
Bacillus
species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore’s inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.
Azospirillum irakense
Produces a Novel Type of Pectate Lyase
Authors: Bekri My Ali , Desair Jos , Keijers Veerle , Proost Paul , Searle-van Leeuwen Marjo , Vanderleyden Jos , vande Broek Ann
Published: December 2019

The
pelA
gene from the N
2
-fixing plant-associated bacterium
Azospirillum irakense
, encoding a pectate lyase, was isolated by heterologous expression in
Escherichia coli
. Nucleotide sequence analysis of the region containing
pelA
indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in
E. coli
at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca
2+
for its activity. Of several divalent cations tested, none could substitute for Ca
2+
. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of
pelA
expression was studied by means of a translational
pelA-gusA
fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition,
pelA
expression was found to be induced by pectin. An
A. irakense pelA
::Tn
5
mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in
A. irakense
.
The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of
Bacillus subtilis
Authors: Tjalsma Harold , van den Dolder Juliëtte , Meijer Wilfried J. J. , Venema Gerard , Bron Sierd , van Dijl Jan Maarten
Published: December 2019

The gram-positive eubacterium
Bacillus subtilis
is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the
B. subtilis
plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with
sipP
. These two genes form a conserved structural module of rolling-circle plasmids from
B. subtilis
. As previously shown for the chromosomal
sipS
and
sipT
genes, the transcription of plasmid-borne copies of
sipP
is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of
sipP
starts at the end of exponential growth, about 2 h earlier than that of
sipS
and
sipT
. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.
Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families
Authors: Hill Katja E. , Marchesi Julian R. , Weightman Andrew J.
Published: December 2019

Dehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial α-halocarboxylic acid (αHA) dehalogenase genes, called group I and group IIdehgenes. The two families are evolutionarily unrelated and together represent almost all of the αHAdehgenes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and IIdehgenes. Amino acid sequences derived from 10 of 11 group Idehpartial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from aRhizobiumsp., had only two of these five residues. Group IIdehgene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinatingl- but notd-2-chloropropionic acid, and derived amino acid sequences for all of the genes exceptdehII°P11showed conservation of previously identified essential residues. Molecular analysis of the twodehfamilies highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group Idehgenes included two putative cryptic or silent genes,dehI°PP3anddehI°17a, produced by different organisms. Group IIdehgenes included two cryptic genes and an active gene,dehIIPP3, that can be switched off and on. All αHA-degrading bacteria so far described wereProteobacteria, a result that may be explained by limitations either in the host range fordehgenes or in isolation methods.
Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the
Bacillus cereus
Group
Authors: Prüß Birgit M. , Francis Kevin P. , von Stetten Felix , Scherer Siegfried
Published: December 2019

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the
Bacillus cereus
group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that
B. cereus
group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.
Molecular and Functional Characterization of the
Rhodopseudomonas palustris
No. 7 Phosphoenolpyruvate Carboxykinase Gene
Authors: Inui Masayuki , Nakata Kaori , Roh Jung Hyeob , Zahn Kenneth , Yukawa Hideaki
Published: December 2019

The
pckA
gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium
Rhodopseudomonas palustris
No. 7. Sequencing of a 2.5-kb chromosomal
Sma
I-
Pst
I fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known
pckA
genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the
pckA
initiation codon and an upstream sequence similar to ς
70
promoters. Studies of a
pckA-lacZ
gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the
pckA
gene was induced in log phase, irrespective of the carbon source. A
R. palustris
No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C
4
-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in
R. palustris
No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.
Intraspecific Diversity of the 23S rRNA Gene and the Spacer Region Downstream in
Escherichia coli
Authors: Antón Ana I. , Martínez-Murcia Antonio J. , Rodríguez-Valera Francisco
Published: December 2019

The molecular microevolution of the 23S rRNA gene (
rrl
) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the
Escherichia coli
ECOR collection. The
rrl
gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (
rrs
) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the
rrs
gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the
rrl
gene seems to be quite similar to that of the
rrs
gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3′ end.
In Vivo Transcription of the
Escherichia coli oxyR
Regulon as a Function of Growth Phase and in Response to Oxidative Stress
Authors: Michán Carmen , Manchado Manuel , Dorado Gabriel , Pueyo Carmen
Published: December 2019

Simultaneous expression of seven genes in
Escherichia coli
was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i)
oxyR
(transcriptional regulator); (ii)
katG
,
dps
,
gorA
, and
ahpCF
(controlled by OxyR); (iii)
sodA
(controlled by SoxRS); and (iv)
trxA
(not related to OxyR or SoxRS). Except for
trxA
, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas
oxyR
,
katG
,
dps
, and
gorA
were activated during exponential growth,
ahpCF
and
sodA
were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for
gorA
and
dps
, respectively. Treatment with H
2
O
2
stimulated expression of the genes (
katG
,
dps
,
ahpCF
, and
gorA
) previously identified as members of the OxyR regulon, except for
oxyR
itself. Induction by H
2
O
2
was a remarkably rapid and reversible process that took place in an OxyR-dependent and ς
S
-independent manner. NaCl induced expression of the genes controlled by OxyR, including the
oxyR
locus. This transcriptional up-regulation was preserved in a strain with the Δ
oxyR
::
kan
mutation, but it was abolished (
ahpCF
) or significantly reduced (
oxyR
and
dps
) in a strain with the
rpoS
::Tn
10
mutation, potentially reflecting positive transcriptional regulation of the
oxyR
regulon by ς
S
. Expression of
trxA
was not increased either by H
2
O
2
stress or by a shift to high-osmolarity conditions.
PhhB, a
Pseudomonas aeruginosa
Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level
Authors: Song Jian , Xia Tianhui , Jensen Roy A.
Published: December 2019

Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in
Pseudomonas aeruginosa
contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in
Escherichia coli
as a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in
trans
. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of
phhB
on
phhA
expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in
E. coli
, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation of
phhB
in
P. aeruginosa
and by comparison of the levels of expression of PhhA in the presence and absence of PhhB in
Escherichia coli
. Experiments using constructs having transcriptional and translational fusions with a
lacZ
reporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of either
l
-tyrosine or
l
-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.
The Periplasmic Nitrate Reductase in
Pseudomonas
sp. Strain G-179 Catalyzes the First Step of Denitrification
Authors: Bedzyk Laura , Wang Tao , Ye Rick W.
Published: December 2019

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in
Pseudomonas
sp. strain G-179, the
nap
gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the
nap
region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap
−
mutant, but that activity could be restored by complementation with the
nap
region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.
IncC of Broad-Host-Range Plasmid RK2 Modulates KorB Transcriptional Repressor Activity In Vivo and Operator Binding In Vitro
Authors: Jagura-Burdzy Grazyna , Kostelidou Kalliope , Pole Jessica , Khare Dheeraj , Jones Anthony , Williams D. Ross , Thomas Christopher M.
Published: December 2019

The
korAB
operon of broad-host-range plasmid RK2 encodes five genes, two of which,
incC
and
korB
, belong to the
parA
and
parB
families, respectively, of genome partitioning functions. Both
korB
and a third gene,
korA
, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of
incC
alone caused rapid displacement of RK2. Using two different reporter systems, we show that
incC
modulates the action of KorB. Using promoter fusions to the reporter gene
xylE
, we show that
incC
potentiates the repression of transcription by
korB
. This modulation of
korB
activity was only observed with
incC1
, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with
incC2
, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His
6
tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.