bluuebunny/crossref_metadata_2025_split huggingface dataset:
Energy Conservation by the H2:Heterodisulfide Oxidoreductase fromMethanosarcina mazeiGö1: Identification of Two Proton-Translocating Segments
Authors: Ide Tina , Bäumer Sebastian , Deppenmeier Uwe 
Published: December 2019

ABSTRACTThe membrane-bound H2:heterodisulfide oxidoreductase system of the methanogenic archaeonMethanosarcina mazeiGö1 catalyzed the H2-dependent reduction of 2-hydroxyphenazine and the dihydro-2-hydroxyphenazine-dependent reduction of the heterodisulfide of HS-CoM and HS-CoB (CoM-S-S-CoB). Washed inverted vesicles of this organism were found to couple both processes with the transfer of protons across the cytoplasmic membrane. The maximal H+/2e−ratio was 0.9 for each reaction. The electrochemical proton gradient (ΔμH+) thereby generated was shown to drive ATP synthesis from ADP plus Pi, exhibiting stoichiometries of 0.25 ATP synthesized per two electrons transported for both partial reactions. ATP synthesis and the generation of ΔμH+were abolished by the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847). The ATP synthase inhibitorN,N′-dicyclohexylcarbodiimide did not affect H+translocation but led to an almost complete inhibition of ATP synthesis and decreased the electron transport rates. The latter effect was relieved by the addition of SF 6847. Thus, the energy-conserving systems showed a stringent coupling which resembles the phenomenon of respiratory control. The results indicate that two different proton-translocating segments are present in the H2:heterodisulfide oxidoreductase system; the first involves the 2-hydroxyphenazine-dependent hydrogenase, and the second involves the heterodisulfide reductase.
Molecular Cloning and Functional Expression of Alternative Oxidase from
Candida albicans
Authors: Huh Won-Ki , Kang Sa-Ouk 
Published: December 2019

ABSTRACT

The
AOX1
gene, which encodes an alternative oxidase, was isolated from the genomic DNA library of
Candida albicans
. The gene encodes a polypeptide consisting of 379 amino acids with a calculated molecular mass of 43,975 Da. The
aox1/aox1
mutant strain did not show cyanide-resistant respiration under normal conditions but could still induce cyanide-resistant respiration when treated with antimycin A. The measurement of respiratory activity and Western blot analysis suggested the presence of another AOX. When
C. albicans AOX1
was expressed in alternative oxidase-deficient
Saccharomyces cerevisiae
, it could confer cyanide-resistant respiration on
S. cerevisiae
.
Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-Dependent Glycerol Dehydratase
Authors: Tobimatsu Takamasa , Kajiura Hideki , Yunoki Michio , Azuma Muneaki , Toraya Tetsuo 
Published: December 2019

ABSTRACT

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of
Klebsiella pneumoniae
, the inactivated enzyme is reactivated in the presence of ATP, Mg
2+
, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them
gdrA
and
gdrB
. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant
Escherichia coli
cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.
Mutations in Catabolite Control Protein CcpA Separating Growth Effects from Catabolite Repression
Authors: Küster Elke , Hilbich Tanja , Dahl Michael K. , Hillen Wolfgang 
Published: December 2019

ABSTRACT

Carbon catabolite repression in
Bacillus megaterium
is mediated by the transcriptional regulator CcpA. A chromosomal deletion of
ccpA
eliminates catabolite repression and reduces the growth rate on glucose. We describe four single-amino-acid mutations in CcpA which separate the growth effect from catabolite repression, suggesting distinct regulatory pathways for these phenotypes.
Isolation and Characterization of Mutations in the
Escherichia coli
Regulatory Protein XapR
Authors: Jørgensen Casper , Dandanell Gert 
Published: December 2019

ABSTRACT

In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in
Escherichia coli
. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from
Klebsiella aerogenes
, are placed close together and are most probably directly involved in binding the inducer xanthosine.
Identification and Complementation of Frameshift Mutations Associated with Loss of Cytadherence in
Mycoplasma pneumoniae
Authors: Fisseha Makda , Göhlmann Hinrich W. H. , Herrmann Richard , Krause Duncan C. 
Published: December 2019

ABSTRACT

Mycoplasma pneumoniae
cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of
hmw2
by Tn
4001
results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in
hmw2
. In this study, the recombinant wild-type
hmw2
allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I
hmw2
allele did not restore cytadherence, consistent with a defect in
hmw2
in this mutant. A frameshift was discovered in different oligoadenine tracts in
hmw2
from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in
hmw2
. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.
Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of
Staphylococcus aureus
Bacteriophage 187
Authors: Loessner Martin J. , Gaeng Susanne , Scherer Siegfried 
Published: December 2019

ABSTRACT

We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique
Staphylococcus aureus
phage 187. The endolysin gene
ply187
encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined
ply187
domains. This enzymatically active N terminus showed convincing amino acid sequence homology to an
N
-acetylmuramoyl-
l
-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of
ply187
and was shown to encode a small, hydrophobic protein of holin-like function. The
hol187
gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in
Escherichia coli
resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected
S. aureus
cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ
S
am mutation in a nonsuppressing
E. coli
HB101 background. Plaque formation by λgt11::
hol187
indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of
ply187
and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected
Staphylococcus
cells.
Illegitimate Recombination Induced by Overproduction of DnaB Helicase in
Escherichia coli
Authors: Yamashita Teruhito , Hanada Katsuhiro , Iwasaki Mihoko , Yamaguchi Hirotaka , Ikeda Hideo 
Published: December 2019

ABSTRACT

Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in
Escherichia coli
. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.
Ni
2+
Transport and Accumulation in
Rhodospirillum rubrum
Authors: Watt Richard K. , Ludden Paul W. 
Published: December 2019

ABSTRACT

The
cooCTJ
gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni
2+
transport assay was used to monitor uptake and accumulation of
63
Ni
2+
into
R. rubrum
and to observe the effect of mutations in the
cooC
,
cooT
, and
cooJ
genes on
63
Ni
2+
transport and accumulation. Cells grown either in the presence or absence of CO transported Ni
2+
with a

K
m

of 19 ± 4 μM and a
V
max
of 310 ± 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the
cooCTJ
genes, either individually or all three genes simultaneously, transported Ni
2+
the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn
2+
, Mg
2+
, Ca
2+
, and Zn
2+
showed no inhibition of
63
Ni
2+
transport but Co
2+
, Cd
2+
, and Cu
2+
inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°C), by protonophores (carbonyl cyanide
m
-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (
N
,
N
′-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni
2+
transport.
63
Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated
63
Ni
2+
accumulation coincided with the appearance of CODH activity in the culture, suggesting that the
63
Ni
2+
was accumulating in CODH. The
cooC
,
cooT
, and
cooJ
genes are required for the increased
63
Ni
2+
accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated
63
Ni
2+
like cells unexposed to CO.
MgATP Binding and Hydrolysis Determinants of NtrC, a Bacterial Enhancer-Binding Protein
Authors: Rombel Irene , Peters-Wendisch Petra , Mesecar Andrew , Thorgeirsson Thorgeir , Shin Yeon-Kyun , Kustu Sydney 
Published: December 2019

ABSTRACT

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of
Salmonella typhimurium
forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the ς
54
-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent

K
d

of unphosphorylated NtrC for MgATPγS is 100 μM at 25°C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 μM at 37°C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of ς
54
-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21

ras

and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn
2+
binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.
Molecular Characterization of Genetic Loci Required for Secretion of Exoproducts in
Burkholderia pseudomallei
Authors: DeShazer David , Brett Paul J. , Burtnick Mary N. , Woods Donald E. 
Published: December 2019

ABSTRACT

Previous studies have demonstrated that
Burkholderia pseudomallei
secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC.
The Thermophilic Yeast
Hansenula polymorpha
Does Not Require Trehalose Synthesis for Growth at High Temperatures but Does for Normal Acquisition of Thermotolerance
Authors: Reinders Anke , Romano Ivano , Wiemken Andres , De Virgilio Claudio 
Published: December 2019

ABSTRACT

The
TPS1
gene from
Hansenula polymorpha
, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of
TPS1
rendered
H. polymorpha
cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells’ ability to acquire thermotolerance.
Amino Acid Residues Involved in the Functional Integrity of
Escherichia coli
Methionine Aminopeptidase
Authors: Chiu Chen-Hsiang , Lee Chao-Zong , Lin Kung-Shih , Tam Ming F. , Lin Lih-Yuan 
Published: December 2019

ABSTRACT

Amino acid residues in the metal-binding and putative substrate-binding sites of
Escherichia coli
methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.
Transport of d-Xylose in Lactobacillus pentosus, Lactobacillus casei, andLactobacillus plantarum: Evidence for a Mechanism of Facilitated Diffusion via the Phosphoenolpyruvate:Mannose Phosphotransferase System
Authors: Chaillou Stéphane , Pouwels Peter H. , Postma Pieter W. 
Published: December 2019

ABSTRACTWe have identified and characterized thed-xylose transport system ofLactobacillus pentosus. Uptake ofd-xylose was not driven by the proton motive force generated by malolactic fermentation and requiredd-xylose metabolism. The kinetics ofd-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparentKmof 8.5 mM and aVmaxof 23 nmol min−1mg of dry weight−1. In two mutants ofL. pentosusdefective in the phosphoenolpyruvate:mannose phosphotransferase system, growth ond-xylose was absent due to the lack ofd-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of theL. curvatus manBgene encoding the cytoplasmic EIIBMancomponent of the EIIMancomplex. The EIIMancomplex is also involved ind-xylose transport inL. caseiATCC 393 andL. plantarum80. These two species could transport and metabolized-xylose after transformation with plasmids which expressed thed-xylose-catabolizing genes ofL. pentosus,xylAB. L. caseiandL. plantarummutants resistant to 2-deoxy-d-glucose were defective in EIIManactivity and were unable to transportd-xylose when transformed with plasmids containing thexylABgenes. Finally, transport ofd-xylose was found to be the rate-limiting step in the growth ofL. pentosusand ofL. plantarumandL. caseiATCC 393 containing plasmids coding for thed-xylose-catabolic enzymes, since the doubling time of these bacteria ond-xylose was proportional to the level of EIIManactivity.
Transformations in Flagellar Structure ofRhodobacter sphaeroides and Possible Relationship to Changes in Swimming Speed
Authors: Armitage Judith P. , Pitta Thomas P. , Vigeant Margot A.-S. , Packer Helen L. , Ford Roseanne M. 
Published: December 2019

ABSTRACT

Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops. Free-swimming R. sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions. DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations. However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix. The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament. In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form. This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation. This form was always significantly longer than the coiled or helical form from which it was derived. The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix. Examination of the three-dimensional swimming pattern showed that R. sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops. The rate of acceleration out of stops was also variable. The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells. The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed.
High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis
Authors: Hare Janelle M. , McDonough Kathleen A. 
Published: December 2019

ABSTRACT

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. ThehmsHFRS genes required for CR binding (Crb+) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb+ phenotype. We constructed a recA mutant strain of Y. pestisKIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flankedpgm locus. Two Pgm-associated phenotypes (Crb+and pesticin sensitivity [Psts]) were used as markers for the presence of the pgm locus in the RecA+KIM10+ and RecA− YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 × 10−3) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb+ phenotype was still lost at a high frequency (4.5 × 10−5), although the loss of the Pstsphenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 × 10−7). These RecA-independent Crb− mutants were caused by mutations in both thehmsHFRS locus and in a newly identified gene,hmsT. Nonpigmented Yersinia pseudotuberculosisand Escherichia coli strains transformed with bothhmsT and hmsHFRS became Crb+. This study demonstrates that in a laboratory culture, the Crb+phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS andhmsT loci.