bluuebunny/crossref_metadata_2025_split huggingface dataset:
Roles of Glutamate Synthase,gltBD, andgltFin Nitrogen Metabolism ofEscherichia coliandKlebsiella aerogenes
Authors: Goss Thomas J. , Perez-Matos Ana , Bender Robert A. 
Published: July 2002

ABSTRACTMutants ofEscherichia coliandKlebsiella aerogenesthat are deficient in glutamate synthase (glutamate-oxoglutarate amidotransferase [GOGAT]) activity have difficulty growing with nitrogen sources other than ammonia. Two models have been proposed to account for this inability to grow. One model postulated an imbalance between glutamine synthesis and glutamine degradation that led to a repression of the Ntr system and the subsequent failure to activate transcription of genes required for the use of alternative nitrogen sources. The other model postulated that mutations ingltBorgltD(which encode the subunits of GOGAT) were polar on a downstream gene,gltF, which is necessary for proper activation of gene expression by the Ntr system. The data reported here show that thegltFmodel is incorrect for three reasons: first, a nonpolargltBand a polargltDmutation ofK. aerogenesboth show the same phenotype; second,K. aerogenesand several other enteric bacteria lack a gene homologous togltF; and third, mutants ofE. coliwhosegltFgene has been deleted show no defect in nitrogen metabolism. The argument that accumulated glutamine represses the Ntr system ingltBorgltDmutants is also incorrect, because these mutants can derepress the Ntr system normally so long as sufficient glutamate is supplied. Thus, we conclude thatgltBorgltDmutants grow slowly on many poor nitrogen sources because they are starved for glutamate. Much of the glutamate formed by catabolism of alternative nitrogen sources is converted to glutamine, which cannot be efficiently converted to glutamate in the absence of GOGAT activity. Finally, GOGAT-deficientE. colicells growing with glutamine as the sole nitrogen source increase their synthesis of the other glutamate-forming enzyme, glutamate dehydrogenase, severalfold, but this is still insufficient to allow rapid growth under these conditions.
Cytochromes
c
550
,
c
552
, and
c
1
in the Electron Transport Network of
Paracoccus denitrificans
: Redundant or Subtly Different in Function?
Authors: Otten Marijke F. , van der Oost John , Reijnders Willem N. M. , Westerhoff Hans V. , Ludwig Bernd , Van Spanning Rob J. M. 
Published: July 2002

ABSTRACT

Paracoccus denitrificans
strains with mutations in the genes encoding the cytochrome
c
550
,
c
552
, or
c
1
and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome
c
mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome
bc
1
complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome
c
550
-deficient mutant did denitrify provided copper was present. The cytochrome
c
552
mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes
c
have multiple tasks in electron transfer. The cytochrome
bc
1
complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes
c
550
and
c
552
and to the
cbb
3
-type oxidase. Cytochrome
c
552
is an electron acceptor both of the cytochrome
bc
1
complex and of amicyanin, as well as a dedicated electron donor to the
aa
3
-type oxidase. Cytochrome
c
550
can accept electrons from the cytochrome
bc
1
complex and from amicyanin, whereas it is also the electron donor to both cytochrome
c
oxidases and to at least the nitrite reductase during denitrification. Deletion of the
c
-type cytochromes also affected the concentrations of remaining cytochromes
c
, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.
Use of Transposon Tn
5367
Mutagenesis and a Nitroimidazopyran-Based Selection System To Demonstrate a Requirement for
fbiA
and
fbiB
in Coenzyme F
420
Biosynthesis by
Mycobacterium bovis
BCG
Authors: Choi Kwang-Pil , Bair Thomas B. , Bae Young-Min , Daniels Lacy 
Published: July 2002

ABSTRACT

Three transposon Tn
5367
mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of
Mycobacterium bovis
strain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F
420
was developed using the nitroimidazopyran-based antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defective in F
420
accumulation. Two mutants that could not make F
420
-5,6 but which made the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in two adjacent homologues of
Mycobacterium tuberculosis
genes, which we have named
fbiA
and
fbiB
for F
420
biosynthesis. Homologues of
fbiA
were found in all seven microorganisms that have been fully sequenced and annotated and that are known to make F
420
.
fbiB
homologues were found in all but one such organism. Complementation of the
fbiA
mutant with
fbiAB
and complementation of the
fbiB
mutant with
fbiB
both restored the F
420
-5,6 phenotype. Complementation of the
fbiA
mutant with
fbiA
or
fbiB
alone did not restore the F
420
-5,6 phenotype, but the
fbiA
mutant complemented with
fbiA
produced F
420
-2,3,4 at levels similar to F
420
-5,6 made by the wild-type strain, but produced much less F
420
-5. These data demonstrate that both genes are essential for normal F
420
-5,6 production and suggest that the
fbiA
mutation has a partial polar effect on
fbiB
. Reverse transcription-PCR data demonstrated that
fbiA
and
fbiB
constitute an operon. However, very low levels of
fbiB
mRNA are produced by the
fbiA
mutant, suggesting that a low-level alternative start site is located upstream of
fbiB
. The specific reactions catalyzed by FbiA and FbiB are unknown, but both function between FO and F
420
-5,6, since FO is made by both mutants.
New Regulatory Gene That Contributes to Control of
Bacteroides thetaiotaomicron
Starch Utilization Genes
Authors: Cho Kyu Hong , Cho Diedre , Wang Gui-Rong , Salyers Abigail A. 
Published: July 2002

ABSTRACT

Bacteroides thetaiotaomicron
uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by
sus
genes, which have been characterized previously. The
sus
structural genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the
sus
structural genes is mediated by SusR, an activator that is encoded by a gene located next to the
sus
structural genes. A strain with a disruption in
susR
cannot grow on starch but can still grow on maltose and maltotriose. A search for transposon-generated mutants that could not grow on maltose and maltotriose unexpectedly located a gene, designated
malR
, which regulates expression of an α-glucosidase not controlled by SusR. Although a disruption in
susR
did not affect expression of the
malR
controlled gene, a disruption in
malR
reduced expression of the
sus
structural genes. Thus, MalR appears to participate with SusR in regulation of the
sus
genes. Results of transcriptional fusion assays and reverse transcription-PCR experiments showed that
malR
is expressed constitutively. Moreover, multiple copies of
malR
provided on a plasmid (5 to 10 copies per cell) more than doubled the amount of α-glucosidase activity in cell extracts. Our results demonstrate that the starch utilization system of
B. thetaiotaomicron
is controlled on at least two levels by the regulatory proteins SusR and MalR.
Hyperactive Glycogen Synthase Mutants of
Saccharomyces cerevisiae
Suppress the
glc7-1
Protein Phosphatase Mutant
Authors: Anderson Catherine , Tatchell Kelly 
Published: July 2002

ABSTRACT

A yeast
glc7-1
mutant expressing a variant of protein phosphatase type 1 fails to accumulate glycogen. This defect is associated with hyperphosphorylated and inactive glycogen synthase, consistent with Glc7p acting directly to dephosphorylate and activate glycogen synthase. To characterize the glycogen synthesis defect of this mutant in more detail, we isolated 26 pseudorevertants of the
glc7-1
mutant. All pseudoreversion events were due to missense mutations in
GSY2
, the gene encoding the major isoform of glycogen synthase. A majority of the mutations responsible for the suppression were in the 3′ end of the gene, corresponding to the phosphorylated COOH terminus of Gsy2p. Phosphorylation of the mutant proteins was reduced, suggesting that they are poor substrates for glycogen synthase kinases. Suppressor mutations outside this domain did not decrease the phosphorylation of the resulting proteins, indicating that these proteins are immune to the regulatory effects of phosphorylation. Since no growth defect has been observed for strains with altered glycogen levels, the relative levels of fitness of
GSY2
mutants that fail to accumulate glycogen and that hyperaccumulate glycogen were assayed by cocultivation experiments. A wild-type strain outcompeted both hypo- and hyperaccumulating strains, suggesting that glycogen levels contribute substantially to the fitness of yeast.
Periplasmic Transit and Disulfide Bond Formation of the Autotransported
Shigella
Protein IcsA
Authors: Brandon Lauren D. , Goldberg Marcia B. 
Published: July 2002

ABSTRACT

The
Shigella
outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal β domain forms a β barrel channel in the outer membrane through which the amino-terminal α domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by
Shigella
, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the
dsbB
gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.
Putative Sigma Factor SigI (YkoZ) of
Bacillus subtilis
Is Induced by Heat Shock
Authors: Zuber Ulrich , Drzewiecki Kathrin , Hecker Michael 
Published: July 2002

ABSTRACT

A
Bacillus subtilis
disruption mutant with a mutation in
sigI
(formerly
ykoZ
) shows a temperature-sensitive growth on agar plates. The transcription of the
sigI
gene is heat shock induced in rich medium but not in minimal medium. Proteome studies revealed a reduced amount of GsiB protein in the
sigI
mutant under heat shock conditions.
Mobile Cytochrome
c
2
and Membrane-Anchored Cytochrome
c
y
Are Both Efficient Electron Donors to the
cbb
3
- and
aa
3
-Type Cytochrome
c
Oxidases during Respiratory Growth of
Rhodobacter sphaeroides
Authors: Daldal Fevzi , Mandaci Sevnur , Winterstein Christine , Myllykallio Hannu , Duyck Kristen , Zannoni Davide 
Published: July 2002

ABSTRACT

We have recently established that the facultative phototrophic bacterium
Rhodobacter sphaeroides
, like the closely related
Rhodobacter capsulatus
species, contains both the previously characterized mobile electron carrier cytochrome
c
2
(cyt
c
2
) and the more recently discovered membrane-anchored cyt
c
y
. However,
R. sphaeroides
cyt
c
y
, unlike that of
R. capsulatus
, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt
bc
1
complex during photosynthetic growth. Nonetheless,
R. sphaeroides
cyt
c
y
can act at least in
R. capsulatus
as an electron carrier between the cyt
bc
1
complex and the
cbb
3
-type cyt
c
oxidase (
cbb
3
-C
ox
) to support respiratory growth. Since
R. sphaeroides
harbors both a
cbb
3
-C
ox
and an
aa
3
-type cyt
c
oxidase (
aa
3
-C
ox
), we examined whether
R. sphaeroides
cyt
c
y
can act as an electron carrier to either or both of these respiratory terminal oxidases.
R. sphaeroides
mutants which lacked either cyt
c
2
or cyt
c
y
and either the
aa
3
-C
ox
or the
cbb
3
-C
ox
were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt
bc
1
complex and the cyt
c
oxidases. They were characterized with respect to growth phenotypes, contents of
a
-,
b
-, and
c
-type cytochromes, cyt
c
oxidase activities, and kinetics of electron transfer mediated by cyt
c
2
or cyt
c
y
. The findings demonstrated that both cyt
c
2
and cyt
c
y
are able to carry electrons efficiently from the cyt
bc
1
complex to either the
cbb
3
-C
ox
or the
aa
3
-C
ox
. Thus, no dedicated electron carrier for either of the cyt
c
oxidases is present in
R. sphaeroides
. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt
bc
1
complex appears to be mediated via the cyt
c
2
-to-
cbb
3
-C
ox
and cyt

c
y

-to-
cbb
3
-C
ox
subbranches. The presence of multiple electron carriers and cyt
c
oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of
R. sphaeroides
are more complex than those of
R. capsulatus
.
Escherichia coli
Ribosome-Associated Protein SRA, Whose Copy Number Increases during Stationary Phase
Authors: Izutsu Kaori , Wada Chieko , Komine Yuriko , Sako Tomoyuki , Ueguchi Chiharu , Nakura Satomi , Wada Akira 
Published: July 2002

ABSTRACT

Protein D has previously been demonstrated to be associated with
Escherichia coli
ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the
E. coli
genetic map, between
ompC
and
sfcA
. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named
sra
. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of
sra
in the stationary phase.
AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in
Rhodobacter capsulatus
Authors: Dong Chen , Elsen Sylvie , Swem Lee R. , Bauer Carl E. 
Published: July 2002

ABSTRACT

Open reading frame
orf192
, which is located immediately upstream of the aerobic repressor gene
crtJ
, was genetically and biochemically demonstrated to code for a second aerobic repressor (AerR) of photosynthesis gene expression in
Rhodobacter capsulatus
. Promoter-mapping studies indicate that
crtJ
has its own promoter but that a significant proportion of
crtJ
expression is promoted by read-through transcription of
orf192
(
aerR
) transcripts through
crtJ
. Disruption of
aerR
resulted in increased photopigment biosynthesis during aerobic growth to a level similar to that of disruption of
crtJ
. Like that reported for CrtJ, β-galactosidase assays of reporter gene expression indicated that disruption of
aerR
resulted in a two- to threefold increase in aerobic expression of the
crtI
and
pucB
operons. However, unlike CrtJ, AerR aerobically represses
puf
operon expression and does not aerobically repress
bchC
expression. Gel mobility shift analysis with purified AerR indicates that AerR does not bind to a
bchC
promoter probe but does bind to the
crtI
,
puc
, and
puf
promoter probes. These results indicate that AerR is a DNA-binding protein that targets genes partially overlapping a subset of genes that are also controlled by CrtJ. We also provide evidence for cooperative binding of AerR and CrtJ to the
puc
promoter region.
Coordination of Ubiquinol Oxidase and Cytochrome
cbb
3
Oxidase Expression by Multiple Regulators in
Rhodobacter capsulatus
Authors: Swem Danielle L. , Bauer Carl E. 
Published: July 2002

ABSTRACT

Rhodobacter capsulatus
utilizes two terminal oxidases for aerobic respiration, cytochrome
cbb
3
and ubiquinol oxidase. To determine the transcription factors involved in terminal oxidase expression,
ccoN
-
lacZ
and
cydA
-
lacZ
protein fusions were assayed in a variety of regulatory mutants. The results of this and previous studies indicate that cytochrome
cbb
3
expression is controlled by
regB
-
regA
,
fnrL
, and
hvrA
and that ubiquinol oxidase expression is controlled by
regB
-
regA
,
fnrL
,
hvrA
,
crtJ
, and
aerR
.
Dam- and OxyR-Dependent Phase Variation of
agn43
: Essential Elements and Evidence for a New Role of DNA Methylation
Authors: Wallecha Anu , Munster Vincent , Correnti Jason , Chan Teresa , van der Woude Marjan 
Published: July 2002

ABSTRACT

Phase variation of the outer membrane protein Ag43 in
E. coli
requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene,
agn43
, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of
agn43
transcription and its regulation. Transcription was initiated from a σ
70
-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of
agn′-lacZ
with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating
agn43
transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the
agn43
regulatory region from −16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the
agn43
regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation
Molecular Organization of Intrinsic Restriction and Modification Genes
Bsu
M of
Bacillus subtilis
Marburg
Authors: Ohshima Hideyuki , Matsuoka Satoshi , Asai Kei , Sadaie Yoshito 
Published: July 2002

ABSTRACT

Transcriptional analysis and disruption of five open reading frames (ORFs),
ydiO
,
ydiP
,
ydiR
,
ydiS
, and
ydjA
, in the prophage 3 region of the chromosome of
Bacillus subtilis
Marburg revealed that they are component genes of the intrinsic
Bsu
M restriction and modification system of this organism. The classical mutant strain RM125, which lacks the restriction and modification system of
B. subtilis
Marburg, lacks the prophage 3 region carrying these five ORFs. These ORFs constitute two operons, the
ydiO-ydiP
operon and the
ydiR
-
ydiS
-
ydjA
operon, both of which are expressed during the logarithmic phase of growth. The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases. The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known. Disruption of the
ydiR-ydiS-ydjA
operon resulted in enhanced transformation by plasmid DNA carrying multiple
Bsu
M target sequences. Disruption of
ydiO
or
ydiP
function requires disruption of at least one of the following genes on the chromosome:
ydiR
,
ydiS
, and
ydjA
. The degrees of methylation of the
Bsu
M target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with
Xho
I (an isoschizomer of
Bsu
M) of DNAs extracted from the disruptant strains. Six
Xho
I (
Bsu
M) sites were examined.
Xho
I digested at the
Xho
I sites in the DNAs from disruptants with disruptions in both operons, while
Xho
I did not digest at the
Xho
I sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the
ydiR-ydiS-ydjA
operon. Therefore, the
ydiO-ydiP
operon and the
ydiR-ydiS-ydjA
operon are considered operons that are responsible for
Bsu
M modification and
Bsu
M restriction, respectively.
Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27
Authors: Lawley Trevor D. , Gilmour Matthew W. , Gunton James E. , Standeven Leah J. , Taylor Diane E. 
Published: July 2002

ABSTRACTThe conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region betweentraHandtrhRwas cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of theoriTregion are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of theoriTregion are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.