bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Autonomously Replicating Transforming Vector for
Sulfolobus solfataricus
Authors: Cannio Raffaele , Contursi Patrizia , Rossi Mosè , Bartolucci Simonetta 
Published: December 2019

ABSTRACT

A plasmid able to transform and to be stably maintained both in
Sulfolobus solfataricus
and in
Escherichia coli
was constructed by insertion into an
E. coli
plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the
hph
(hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in
Sulfolobus
was increased by exposure of the cells to mitomycin C.
Appropriate Expression of Filamentous Phage f1 DNA Replication Genes II and X Requires RNase E-Dependent Processing and Separate mRNAs
Authors: Kokoska Robert J. , Steege Deborah A. 
Published: December 2019

ABSTRACT

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected
Escherichia coli
hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on
rne
, the gene encoding the only
E. coli
endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.
Establishment of Prespore-Specific Gene Expression in
Bacillus subtilis
: Localization of SpoIIE Phosphatase and Initiation of Compartment-Specific Proteolysis
Authors: Lewis Peter J. , Wu Ling Juan , Errington Jeffery 
Published: December 2019

ABSTRACT

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in
Bacillus subtilis
. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor ς
E
and the other dependent on the prespore-specific sigma factor ς
F
.
A Complex Pattern of Traveling Stripes Is Produced by Swimming Cells of
Bacillus subtilis
Authors: Mendelson Neil H. , Lega Joceline 
Published: December 2019

ABSTRACT

Motile cells of
Bacillus subtilis
inadvertently escaped from the surface of an agar disk that was surrounded by a fluid growth medium and formed a migrating population in the fluid. When viewed from above, the population appeared as a cloud advancing unidirectionally into the fresh medium. The cell population became spontaneously organized into a series of stripes in a region behind the advancing cloud front. The number of stripes increased progressively until a saturation value of stripe density per unit area was reached. New stripes arose at a fixed distance behind the cloud front and also between stripes. The spacing between stripes underwent changes with time as stripes migrated towards and away from the cloud front. The global pattern appeared to be stretched by the advancing cloud front. At a time corresponding to approximately two cell doublings after pattern formation, the pattern decayed, suggesting that there is a maximum number of cells that can be maintained within the pattern. Stripes appear to consist of high concentrations of cells organized in sinking columns that are part of a bioconvection system. Their behavior reveals an interplay between bacterial swimming, bioconvection-driven fluid motion, and cell concentration. A mathematical model that reproduces the development and dynamics of the stripe pattern has been developed.
Purification and Properties of the F
1
F
o
ATPase of
Ilyobacter tartaricus
, a Sodium Ion Pump
Authors: Neumann Sandra , Matthey Ulrich , Kaim Georg , Dimroth Peter 
Published: December 2019

ABSTRACT

The ATPase of
Ilyobacter tartaricus
was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F
1
F
o
ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, ɛ, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na
+
-translocating ATPase of
Propionigenium modestum
. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na
+
ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na
+
or Li
+
ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na
+
, Li
+
, or H
+
. Proton transport was specifically inhibited by Na
+
or Li
+
ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the
I. tartaricus
ATPase as a new member of the family of FS-ATPases, which use Na
+
as the physiological coupling ion for ATP synthesis.
Gene Replacement Analysis of the
Streptomyces virginiae barA
Gene Encoding the Butyrolactone Autoregulator Receptor Reveals that BarA Acts as a Repressor in Virginiamycin Biosynthesis
Authors: Nakano Hiroko , Takehara Emio , Nihira Takuya , Yamada Yasuhiro 
Published: December 2019

ABSTRACT

Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of
Streptomyces
species, act as a primary signal in
Streptomyces virginiae
to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two
barA
mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp
Eco
T14I fragment of
barA
was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type
S. virginiae
on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in
S. virginiae
. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain NH2, an internal
Eco
NI-
Sma
I fragment of
barA
was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutated
barA
genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.
Expression of the
bglH
Gene of
Lactobacillus plantarum
Is Controlled by Carbon Catabolite Repression
Authors: Marasco Rosangela , Muscariello Lidia , Varcamonti Mario , De Felice Maurilio , Sacco Margherita 
Published: December 2019

ABSTRACT

A newly identified
bglH
gene coding for a phospho-β-glucosidase of
Lactobacillus plantarum
was isolated and expressed in
Escherichia coli
. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The
bglH
gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in
L. plantarum
cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of
bglH
expression.
The A
1
A
o
ATPase from
Methanosarcina mazei
: Cloning of the 5′ End of the
aha
Operon Encoding the Membrane Domain and Expression of the Proteolipid in a Membrane-Bound Form in
Escherichia coli
Authors: Ruppert Claudia , Wimmers Sönke , Lemker Thorsten , Müller Volker 
Published: December 2019

ABSTRACT

Three additional ATPase genes, clustered in the order
ahaH
,
ahaI
, and
ahaK
, were found upstream of the previously characterized genes
ahaECFABDG
coding for the archaeal A
1
A
o
ATPase from
Methanosarcina mazei
.
ahaH
, the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis revealed that the clusters
ahaHIK
and
ahaECFABDG
are transcribed as one message. AhaH is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in
Methanobacterium thermoautotrophicum
and
Methanococcus jannaschii
. AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane α helices. It is similar to the 100-kDa polypeptide of V
1
V
o
ATPases and is therefore suggested to participate in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane α helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from
M. thermoautotrophicum
and
M. jannaschii
, respectively.
ahaK
is expressed in
Escherichia coli
, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria. This is the first report on the expression and incorporation into
E. coli
lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase.
Does Disparate Occurrence of Autoregulatory Programmed Frameshifting in Decoding the Release Factor 2 Gene Reflect an Ancient Origin with Loss in Independent Lineages?
Authors: Persson Britt C. , Atkins John F. 
Published: December 2019

ABSTRACT

In
Escherichia coli
an autoregulatory mechanism of programmed ribosomal frameshifting governs the level of polypeptide chain release factor 2. From an analysis of 20 sequences of genes encoding release factor 2, we infer that this frameshift mechanism was present in a common ancestor of a large group of bacteria and has subsequently been lost in three independent lineages.
The Glycyl Radical Enzyme TdcE Can Replace Pyruvate Formate-Lyase in Glucose Fermentation
Authors: Sawers Gary , Heßlinger Christian , Muller Nathalie , Kaiser Manuela 
Published: December 2019

ABSTRACT

Mutants of
Escherichia coli
unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the
pfl
gene from
Clostridium butyricum
, we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an
E. coli pfl
mutation. The clostridial gene encoded a basic 14.5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn
554
from
Staphylococcus aureus
. Our studies revealed that the clostridial TcbC protein activated the transcription of the
E. coli tdcABCDEFG
operon, which encodes an anaerobic
l
-threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when
E. coli
grows in the absence of catabolite-repressing sugars and in the presence of
l
-threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the
tdc
operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Heßlinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477–492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to
pfl
mutants.
Isolation and Characterization of
Bacillus subtilis sigB
Operon Mutations That Suppress the Loss of the Negative Regulator RsbX
Authors: Smirnova Natalya , Scott Janelle , Voelker Uwe , Haldenwang W. G. 
Published: December 2019

ABSTRACT

ς
B
, a transcription factor that controls the
Bacillus subtilis
general stress response regulon, is activated by either a drop in intracellular ATP or exposure to environmental stress. RsbX, one of seven ς
B
regulators (Rsb proteins) whose genes are cotranscribed with ς
B
, is a negative regulator in the stress-dependent activation pathway. To better define the interactions that take place among the Rsb proteins, we analyzed
sigB
operon mutations which suppress the high-level ς
B
activity that normally accompanies the loss of RsbX. Each of these mutations was in one of three genes (
rsbT
, -
U
, and -
V
) which encode positive regulators of ς
B
, and they all defined amino acid changes which either compromised the activities of the mutant Rsbs or affected their ability to accumulate. ς
B
activity remained inducible by ethanol in several of the RsbX
−
suppressor strains. This finding supports the notion that RsbX is not needed as the target for ς
B
activation by at least some stresses. ς
B
activity in several RsbX
−
strains with suppressor mutations in
rsbT
or -
U
was high during growth and underwent a continued, rather than a transient, increase following stress. Thus, RsbX is likely responsible for maintaining low ς
B
activity during balanced growth and for reestablishing ς
B
activity at prestress levels following induction. Although RsbX likely participates in limiting the ς
B
induction response, a second mechanism for curtailing unrestricted ς
B
activation was suggested by the ς
B
induction profile in two suppressor strains with mutations in
rsbV
. ς
B
activity in these mutants was stress inducible but transient, even in the absence of RsbX.
Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis
Authors: Abouhamad Walid N. , Bray Dennis , Schuster Martin , Boesch Kristin C. , Silversmith Ruth E. , Bourret Robert B. 
Published: December 2019

ABSTRACT

Escherichia coli
responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except
cheA
and
cheY
resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the
che
genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.
Species-Dependent Phenotypes of Replication-Temperature-Sensitive
trfA
Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of
trfA
Expression
Authors: Karunakaran Ponniah , Blatny Janet Martha , Ertesvåg Helga , Valla Svein 
Published: December 2019

ABSTRACT

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four
trfA
mutants temperature sensitive for replication in
Pseudomonas aeruginosa
. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in
Escherichia coli
. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in
P. aeruginosa
, and the effect of the neutral mutation was caused by its ability to strongly reduce the
trfA
expression level. The double mutant and mutant 163C could not be stably maintained in
E. coli
, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the
trfA
expression level also in
E. coli
. The glycine 85 codon in
trfA
mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type
trfA
was expressed at much lower levels in
E. coli
than in
P. aeruginosa
, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The
E. coli lacZ
gene was translationally fused at the 3′ end and internally in
trfA
, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.
Identification of an Intragenic Ribosome Binding Site That Affects Expression of the
uncB
Gene of the
Escherichia coli
Proton-Translocating ATPase (
unc
) Operon
Authors: Matten Sharlene R. , Schneider Thomas D. , Ringquist Steven , Brusilow William S. A. 
Published: December 2019

ABSTRACT

The
uncB
gene codes for the a subunit of the F
o
proton channel sector of the
Escherichia coli
F
1
F
o
ATPase. Control of expression of
uncB
appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997–3011, 1982) identified a potential ribosome binding site within the
uncB
reading frame preceding a five-codon reading frame which is shifted one base relative to the
uncB
reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an
uncB
′-′
lacZ
fusion gene containing most of
uncB
. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of
lacZ
to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of
uncB
is reduced by translational frameshifting and/or a translational false start at this site within the
uncB
reading frame.
Genetic Analysis of Dioxin Dioxygenase of
Sphingomonas
sp. Strain RW1: Catabolic Genes Dispersed on the Genome
Authors: Armengaud Jean , Happe Birgitta , Timmis Kenneth N. 
Published: December 2019

ABSTRACT

The dioxin dioxygenase of
Sphingomonas
sp. strain RW1 activates dibenzo-
p
-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The
dxnA1
and
dxnA2
cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of
dxnA1A2
is modulated according to the available carbon source, expression of the
dbfB
gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of
dxnA1A2
but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of
Sphingomonas
sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the α and β subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in
Escherichia coli
cells containing the cloned
dxnA1A2
gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-
p
-dioxin was obtained. The dioxin dioxygenase was not active in
E. coli
cells coexpressing a class IIB electron supply system. In the course of the isolation of the
dxnA1
and
dxnA2
cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genus
Sphingomonas
, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as
Pseudomonas
species.