bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic and Functional Analysis of the Styrene Catabolic Cluster of
Pseudomonas
sp. Strain Y2
Authors: Velasco Ana , Alonso Sergio , García José L. , Perera J. , Díaz Eduardo 
Published: December 2019

ABSTRACT

The chromosomal region of
Pseudomonas
sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes,
styABCD
, and two regulatory genes,
stySR
, were identified. This gene cluster when transferred to
Escherichia coli
W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source. Genes
styABCD
are homologous to those encoding the styrene upper catabolic pathway in
Pseudomonas fluorescens
ST. Northern blot analyses have confirmed that genes
styABCD
constitute a transcription unit. The transcription start site of the
sty
operon was mapped 33 nucleotides upstream of the
styA
translational start codon. The
styS
and
styR
genes, which form an independent transcriptional unit, are located upstream of the
styABCD
operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The
styS
gene product is homologous to histidine kinase proteins, whereas the
styR
gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains. Expression of the catabolic operon decreased significantly in the absence of the
stySR
genes and was restored when the
stySR
genes were provided in
trans
in the presence of styrene, suggesting that the
stySR
system behaves as a styrene-inducible positive regulator of the
styABCD
operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the
styS
gene. This activity was found to be induced in
Pseudomonas
sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of
Pseudomonas
sp. strain Y2.
Helicobacter pylori porCDABandoorDABCGenes Encode Distinct Pyruvate:Flavodoxin and 2-Oxoglutarate:Acceptor Oxidoreductases Which Mediate Electron Transport to NADP
Authors: Hughes Nicky J. , Clayton Chris L. , Chalk Peter A. , Kelly David J. 
Published: December 2019

ABSTRACTHelicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953–3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype ofH. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. TheH. pylori por,oor, andfldAgenes were cloned and sequenced. The deducedporgene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophileHalobacterium halobium. BothporDandoorDencode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes inH. pylori, as insertion inactivation ofporBandoorAappeared to be lethal.
Regulation of Proteolysis of the Stationary-Phase Sigma Factor RpoS
Authors: Zhou Yanning , Gottesman Susan 
Published: December 2019

ABSTRACT

RpoS, the stationary-phase sigma factor of
Escherichia coli
, is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation in
clpP
, cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.
Genetic Analysis of the
nuo
Locus, Which Encodes the Proton-Translocating NADH Dehydrogenase in
Escherichia coli
Authors: Falk-Krzesinski Holly J. , Wolfe Alan J. 
Published: December 2019

ABSTRACT

Complex I (EC
1.6.99.3
) of the bacterium
Escherichia coli
is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the
E. coli
enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which
E. coli
cells regulate the expression of
nuo
and the assembly of complex I, we undertook a genetic analysis of the
nuo
locus, which encodes the 14 Nuo subunits comprising
E. coli
complex I. Here we present the results of studies, performed on an isogenic collection of
nuo
mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of
nuo
expression and/or the assembly of complex I.
Enzyme Specificity of 2-Nitrotoluene 2,3-Dioxygenase from
Pseudomonas
sp. Strain JS42 Is Determined by the C-Terminal Region of the α Subunit of the Oxygenase Component
Authors: Parales Juanito V. , Parales Rebecca E. , Resnick Sol M. , Gibson David T. 
Published: December 2019

ABSTRACT

Biotransformations with recombinant
Escherichia coli
expressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from
Pseudomonas
sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from
Burkholderia
sp. strain DNT (formerly
Pseudomonas
sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISPα) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISPβ) was shown to play no role in determining the specificities of these dioxygenases.
Identification of
dnaX
as a High-Copy Suppressor of the Conditional Lethal and Partition Phenotypes of the
parE10
Allele
Authors: Levine Cindy , Marians Kenneth J. 
Published: December 2019

ABSTRACT

Termination of DNA replication, complete topological unlinking of the parental template DNA strands, partition of the daughter chromosomes, and cell division follow in an ordered and interdependent sequence during normal bacterial growth. In
Escherichia coli
, topoisomerase IV (Topo IV), encoded by
parE
and
parC
, is responsible for decatenation of the two newly formed chromosomes. In an effort to uncover the pathway of information flow between the macromolecular processes that describe these events, we identified
dnaX
, encoding the τ and γ subunits of the DNA polymerase III holoenzyme, as a high-copy suppressor of the temperature-sensitive phenotype of the
parE10
allele. We show that suppression derives from overexpression of the γ, but not the τ, subunit of the holoenzyme and that the partition defect of
parE10
cells is nearly completely reverted at the nonpermissive temperature as well. These observations suggest a possible association between Topo IV and the replication machinery.
HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in
Mycoplasma pneumoniae
Authors: Hahn Tae-Wook , Willby Melisa J. , Krause Duncan C. 
Published: December 2019

ABSTRACT

Mycoplasma pneumoniae
proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in
hmw1
and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end of
p30
. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type
p30
allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type
M. pneumoniae
cells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant
p30
had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type
hmw1
allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an
hmw1
gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.
Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the
Escherichia coli
Phosphate Regulon
Authors: Haldimann Andreas , Daniels Larry L. , Wanner Barry L. 
Published: December 2019

ABSTRACT

Escherichia coli
genes regulated by environmental inorganic phosphate (P
i
) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the P
i
-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated

P
araB

promoter or the rhamnose-regulated

P
rhaB

promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of

P
araB

or

P
rhaB

fusions and for recombination of them onto the
E. coli
chromosome at the
araCBAD
or
rhaRSBAD
locus, respectively. Using strains carrying such single-copy fusions, we showed that a

P
rhaB

fusion is more tightly regulated than a

P
araB

fusion in that a

P
rhaB
-phoR

+
fusion but not a

P
araB
-phoR

+
fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both

P
araB
-phoB

+
and

P
rhaB
-phoB

+
fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise Δ
phoU
mutations. However, we unexpectedly found that such Δ
phoU
mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797–6809, 1993). They also readily give rise to compensatory mutants with lesions in
phoB
,
phoR
, or a
pst
gene, making their study particularly difficult. Here we found that, by using

P
araB
-phoB

+
,

P
rhaB
-phoB

+
, or

P
rhaB
-phoR

+
fusions, we were able to overcome the extremely deleterious growth defect of a Pst
+
Δ
phoU
mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.
Localization of Cell Division Protein FtsK to the
Escherichia coli
Septum and Identification of a Potential N-Terminal Targeting Domain
Authors: Yu Xuan-chuan , Tran Anthony H. , Sun Qin , Margolin William 
Published: December 2019

ABSTRACT

Escherichia coli
cell division protein FtsK is a homolog of
Bacillus subtilis
SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in
E. coli
cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the
ftsK44
mutation in
trans
, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of
ftsK44
, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and
ftsI
mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in
ftsZ
mutant filaments. In
ftsK44
mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the
ftsK44
mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.
Characterization of IS
1515
, a Functional Insertion Sequence in
Streptococcus pneumoniae
Authors: Muñoz Rosario , López Rubens , García Ernesto 
Published: December 2019

ABSTRACT

We describe the characterization of a new insertion sequence, IS
1515
, identified in the genome of
Streptococcus pneumoniae
I41R, an unencapsulated mutant isolated many years ago (R. Austrian, H. P. Bernheimer, E. E. B. Smith, and G. T. Mills, J. Exp. Med. 110:585–602, 1959). A copy of this element located in the
cap1E
I41R
gene was sequenced. The 871-bp-long IS
1515
element possesses 12-bp perfect inverted repeats and generates a 3-bp target duplication upon insertion. The IS encodes a protein of 271 amino acid residues similar to the putative transposases of other insertion sequences, namely IS
1381
from
S. pneumoniae
, ISL
2
from
Lactobacillus helveticus
, IS
702
from the cyanobacterium
Calothrix
sp. strain PCC 7601, and IS
112
from
Streptomyces albus
G. IS
1515
appears to be present in the genome of most type 1 pneumococci in a maximum of 13 copies, although it has also been found in the chromosome of pneumococcal isolates belonging to other serotypes. We have found that the unencapsulated phenotype of strain I41R is the result of both the presence of an IS
1515
copy and a frameshift mutation in the
cap1E
I41R
gene. Precise excision of the IS was observed in the type 1 encapsulated transformants isolated in experiments designed to repair the frameshift. These results reveal that IS
1515
behaves quite differently from other previously described pneumococcal insertion sequences. Several copies of IS
1515
were also able to excise and move to another locations in the chromosome of
S. pneumoniae
. To our knowledge, this is the first report of a functional IS in pneumococcus.
Loss of the Plasma Membrane-Bound Protein Gas1p in
Saccharomyces cerevisiae
Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity
Authors: Ram Arthur F. J. , Kapteyn Johan C. , Montijn Roy C. , Caro L. Heleen P. , Douwes Jeroen E. , Baginsky Walter , Mazur Paul , van den Ende Herman , Klis Frans M. 
Published: December 2019

ABSTRACT

Deletion of
GAS1/GGP1/CWH52
results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that
gas1
Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.
A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis
Authors: Heath Richard J. , Rock Charles O. 
Published: December 2019

ABSTRACT

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX
4
D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the
sn
-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of
Escherichia coli
. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX
4
D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.
The S-Layer Proteins of Two
Bacillus stearothermophilus
Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition
Authors: Egelseer Eva Maria , Leitner Karl , Jarosch Marina , Hotzy Christoph , Zayni Sonja , Sleytr Uwe B. , Sára Margit 
Published: December 2019

ABSTRACT

Two
Bacillus stearothermophilus
wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from
B. stearothermophilus
PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from
B. stearothermophilus
ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from
B. stearothermophilus
ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from
B. stearothermophilus
PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from
B. stearothermophilus
wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.