bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Evolution of
Mycoplasma capricolum
subsp.
capripneumoniae
Strains, Based on Polymorphisms in the 16S rRNA Genes
Authors: Pettersson Bertil , Bölske Göran , Thiaucourt François , Uhlén Mathias , Johansson Karl-Erik
Published: December 2019

Mycoplasma capricolum
subsp.
capripneumoniae
belongs to the so-called
Mycoplasma mycoides
cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the
M. mycoides
cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of
M. capricolum
subsp.
capripneumoniae
of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the
M. capricolum
subsp.
capripneumoniae
strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of
M. capricolum
subsp.
capripneumoniae
may be used as epidemiological markers for CCPP.
Gas Vesicle Genes Identified in
Bacillus megaterium
and Functional Expression in
Escherichia coli
Authors: Li Ning , Cannon Maura C.
Published: December 2019

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (
gvp
) from
Bacillus megaterium
VT1660 and their functional expression in
Escherichia coli
. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of
E. coli
strains that carry this
gvp
gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-constrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In
B. megaterium
, the
gvp
region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and
gvpA
, -
P
, -
Q
, -
B
, -
R
, -
N
, -
F
, -
G
, -
L
, -
S
, -
K
, -
J
, -
T
, and -
U
, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in
E. coli
. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.
Division Planes Alternate in Spherical Cells of
Escherichia coli
Authors: Begg K. J. , Donachie W. D.
Published: December 2019

In the spherical cells of
Escherichia coli rodA
mutants, division is initiated at a single point, from which a furrow extends progressively around the cell. Using “giant”
rodA ftsA
cells, we confirmed that each new division furrow is initiated at the midpoint of the previous division plane and runs perpendicular to it.
Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of
Streptomyces granaticolor
Authors: Nádvorník Richard , Vomastek Tomáš , Janeček Jiří , Techniková Zuzana , Branny Pavel
Published: December 2019

A 4.2-kb
Sph
I-
Bam
HI fragment of chromosomal DNA from
Streptomyces granaticolor
was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The
pkg2
gene was overexpressed in
Escherichia coli
, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the
pkg2
gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.
Mutations Affecting the Ability of the
Escherichia coli
UmuD′ Protein To Participate in SOS Mutagenesis
Authors: Ohta Toshihiro , Sutton Mark D. , Guzzo Angelina , Cole Shannon , Ferentz Ann E. , Walker Graham C.
Published: December 2019

The products of the SOS-regulated
umuDC
operon are required for most UV and chemical mutagenesis in
Escherichia coli
, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable
umuD
mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190–7194, 1990). To identify elements of the UmuD′ protein necessary for its role in translesion synthesis, we began with
umuD
′, a modified form of the
umuD
gene that directly encodes the UmuD′ protein, and obtained missense
umuD
′ mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD′
2
homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded β-sheet and appears to interfere with UmuD′
2
homodimer formation indirectly by affecting the structure of the UmuD′ monomer. When expressed from a multicopy plasmid, the L40R
umuD
′ mutant gene exhibited a dominant negative effect on a chromosomal
umuD
+
gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD′ function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal β-strand and results in a mutant UmuD′ protein that exhibits a strongly dominant negative effect on UV mutagenesis in a
umuD
+
strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD′
2
homodimer, which are mobile in solution.
pSa Causes Oncogenic Suppression of
Agrobacterium
by Inhibiting VirE2 Protein Export
Authors: Lee Lan-Ying , Gelvin Stanton B. , Kado Clarence I.
Published: December 2019

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the
osa
gene of the plasmid pSa can suppress crown gall tumorigenesis incited by
Agrobacterium tumefaciens
. Neither T-DNA processing nor
vir
(virulence) gene induction is affected by the presence of
osa
in the bacterium. We used
Arabidopsis thaliana
root segments and tobacco leaf discs to demonstrate that Osa inhibits
A. tumefaciens
from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable β-glucuronidase (GUS) expression. When
A. tumefaciens
contained
osa
, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of
Nicotiana benthamiana
by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an
A. tumefaciens virE2
mutant (the T-DNA donor strain) by an
A. tumefaciens
strain lacking T-DNA but containing a wild-type
virE2
gene (the VirE2 donor strain) was blocked when
osa
was present in the VirE2 donor strain, but not when
osa
was present in the T-DNA donor strain. These data indicate that
osa
inhibits VirE2 protein, but not T-DNA export from
A. tumefaciens
. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of
Datura stramonium
plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an
A. tumefaciens virE2
mutant carrying
osa
confirmed that oncogenic suppression by
osa
does not occur by blocking T-DNA transfer. Overexpression of
virB9
,
virB10
, and
virB11
in
A. tumefaciens
did not overcome oncogenic suppression by
osa
. The finding that the expression of the
osa
gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by
osa
may differ from that of the IncQ plasmid RSF1010.
Evidence that the Extracytoplasmic Function Sigma Factor ς
E
Is Required for Normal Cell Wall Structure in
Streptomyces coelicolor
A3(2)
Authors: Paget Mark S. B. , Chamberlin Leony , Atrih Abdelmadjid , Foster Simon J. , Buttner Mark J.
Published: December 2019

The
sigE
gene of
Streptomyces coelicolor
A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed
sigE
deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from
Bacillus subtilis
. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The
sigE
mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that
sigE
is required for normal cell wall structure. The role of ς
E
was further investigated by analyzing the expression of
hrdD
, which is partially
sigE
dependent. The
hrdD
gene, which encodes the ς
HrdD
subunit of RNA polymerase, is transcribed from two promoters,
hrdDp
1
and
hrdDp
2
, both similar to promoters recognized by other ECF sigma factors. The activities of
hrdDp
1
and
hrdDp
2
were reduced 20- and 3-fold, respectively, in
sigE
mutants, although only
hrdDp
1
was recognized by Eς
E
in vitro. Growth on media deficient in magnesium caused the induction of both
hrdD
promoters in a
sigE
-dependent manner.
Divalent Cation Block of Inward Currents and Low-Affinity K
+
Uptake in
Saccharomyces cerevisiae
Authors: Roberts Stephen K. , Fischer Marc , Dixon Graham K. , Sanders Dale
Published: December 2019

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a
trk1Δ trk2Δ
strain of
Saccharomyces cerevisiae
which lacks high- and moderate-affinity K
+
uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K
+
and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K
+
with Rb
+
, Cs
+
, or Na
+
only slightly modulated the magnitude of the inward current, whereas replacement with Li
+
reduced the inward current by approximately 50%, and tetraethylammonium (TEA
+
) and choline were relatively impermeant. The inward current was blocked by extracellular Ca
2+
and Mg
2+
with apparent

K
i

s (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K
+
increased the magnitude of the inward current independently of the electrochemical driving force for K
+
influx, consistent with regulation of the inward current by cytosolic K
+
. Uptake of
86
Rb
+
by intact
trk1Δ trk2Δ
cells was inhibited by extracellular Ca
2+
with a

K
i

within the range observed for the inward current. Furthermore, increasing extracellular Ca
2+
from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.
Transduction-Like Gene Transfer in the Methanogen
Methanococcus voltae
Authors: Bertani Giuseppe
Published: December 2019

Strain PS of
Methanococcus voltae
(a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (
voltae
transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the
M. voltae
cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 × 10
−5
(BES resistance) to a maximum of 10
−3
(histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the
Alu
I restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of
M. voltae
.
Alterations in Protein Expression Caused by the
hha
Mutation in
Escherichia coli
: Influence of Growth Medium Osmolarity
Authors: Balsalobre Carlos , Johansson Jörgen , Uhlin Bernt Eric , Juárez Antonio , Muñoa Francisco J.
Published: December 2019

The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the
hha
mutation on the overall protein pattern of
Escherichia coli
cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the
hha
mutation. The number of proteins whose expression was altered in
hha
cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the
hha
allele, both OmpA and protein IIA
Glc
of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of the
lacZ
gene was also modified in
hha
mutants. This was the case, suggesting that at least some of the pleiotropic effects of the
hha
mutation could be caused by its effect on the catabolite repression system.
The A Modules of the
Azotobacter vinelandii
Mannuronan-C-5-Epimerase AlgE1 Are Sufficient for both Epimerization and Binding of Ca
2+
Authors: Ertesvåg Helga , Valla Svein
Published: December 2019

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

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

(His)
6

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

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

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

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

K
m

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

K
m

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

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

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

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

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

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

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

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

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

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

The glutamyl-tRNA synthetase (
gltX
) gene from
Pseudomonas aeruginosa
was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive
gltX
mutation of
Escherichia coli
JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from
Rhizobium meliloti
.
Nitrate and Nitrite Control of Respiratory Nitrate Reduction in Denitrifying
Pseudomonas stutzeri
by a Two-Component Regulatory System Homologous to NarXL of
Escherichia coli
Authors: Härtig Elisabeth , Schiek Ulrike , Vollack Kai-Uwe , Zumft Walter G.
Published: December 2019

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium
Pseudomonas stutzeri
ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the
narG
operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [
Pichia
] and plant [
Nicotiana
] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of
narG
,
narK
, and
dnrE
but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of
narG
,
narK
, and
dnrE
carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for
Escherichia coli
. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of
P. stutzeri
for selective regulation of nitrate respiration.
Functioning of DcuC as the C
4
-Dicarboxylate Carrier during Glucose Fermentation by
Escherichia coli
Authors: Zientz Evelyn , Janausch Ingo G. , Six Stephan , Unden Gottfried
Published: December 2019

The
dcuC
gene of
Escherichia coli
encodes an alternative C
4
-dicarboxylate carrier (DcuC) with low transport activity. The expression of
dcuC
was investigated.
dcuC
was expressed only under anaerobic conditions; nitrate and fumarate caused slight repression and stimulation of expression, respectively. Anaerobic induction depended mainly on the transcriptional regulator FNR. Fumarate stimulation was independent of the fumarate response regulator DcuR. The expression of
dcuC
was not significantly inhibited by glucose, assigning a role to DcuC during glucose fermentation. The inactivation of
dcuC
increased fumarate-succinate exchange and fumarate uptake by DcuA and DcuB, suggesting a preferential function of DcuC in succinate efflux during glucose fermentation. Upon overexpression in a
dcuC
promoter mutant (
dcuC
*), DcuC was able to compensate for DcuA and DcuB in fumarate-succinate exchange and fumarate uptake.
Bacterioferritin A Modulates Catalase A (KatA) Activity and Resistance to Hydrogen Peroxide in
Pseudomonas aeruginosa
Authors: Ma Ju-Fang , Ochsner Urs A. , Klotz Martin G. , Nanayakkara Vagira K. , Howell Michael L. , Johnson Zaiga , Posey James E. , Vasil Michael L. , Monaco John J. , Hassett Daniel J.
Published: December 2019

We have cloned a 3.6-kb genomic DNA fragment from
Pseudomonas aeruginosa
harboring the
rpoA
,
rplQ
,
katA
, and
bfrA
genes. These loci are predicted to encode, respectively, (i) the α subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome
b
1
or
b
557
). Our goal was to determine the contributions of KatA and BfrA to the resistance of
P. aeruginosa
to hydrogen peroxide (H
2
O
2
). When provided on a multicopy plasmid, the
P. aeruginosa katA
gene complemented a catalase-deficient strain of
Escherichia coli
. The
katA
gene was found to contain two translational start codons encoding a heteromultimer of ∼160 to 170 kDa and having an apparent

K
m

for H
2
O
2
of 44.7 mM. Isogenic
katA
and
bfrA
mutants were hypersusceptible to H
2
O
2
, while a
katA bfrA
double mutant demonstrated the greatest sensitivity. The
katA
and
katA bfrA
mutants possessed no detectable catalase activity. Interestingly, a
bfrA
mutant expressed only ∼47% the KatA activity of wild-type organisms, despite possessing wild-type
katA
transcription and translation. Plasmids harboring
bfrA
genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the
bfrA
mutant. RNase protection assays revealed that
katA
and
bfrA
are on different transcripts, the levels of which are increased by both iron and H
2
O
2
. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the
bfrA
,
katA
, and
katA bfrA
mutants relative to wild-type bacteria. Our results suggest that
P. aeruginosa
BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H
2
O
2
.
Biochemical and Molecular Characterization of the
Bacillus subtilis
Acetoin Catabolic Pathway
Authors: Huang Min , Oppermann-Sanio Fred Bernd , Steinbüchel Alexander
Published: December 2019

A recent study indicated that
Bacillus subtilis
catabolizes acetoin by enzymes encoded by the
acu
gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259–271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by
aco
gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering
acoA
and
acoB
of the AoDH ES from
Clostridium magnum
, genomic fragments from
B. subtilis
harboring
acoA
,
acoB
,
acoC
,
acoL
, and
acoR
homologous genes were identified, and some of them were functionally expressed in
E. coli
. Furthermore,
acoA
was inactivated in
B. subtilis
by disruptive mutagenesis; these mutants were impaired to express PP
i
-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in
B. subtilis
by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described
acu
genes obscure.
Coordinate Transcriptional Control in the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Authors: Haseltine Cynthia , Montalvo-Rodriguez Rafael , Bini Elisabetta , Carl Audrey , Blum Paul
Published: December 2019

The existence of a global gene regulatory system in the hyperthermophilic archaeon
Sulfolobus solfataricus
is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an α-glucosidase (
malA
), a β-glycosidase (
lacS
), and an α-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-α-glucosidase and anti-β-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of
malA
and
lacS
mRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in
lacS
gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. Since
lacS
mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response.