bluuebunny/crossref_metadata_2025_split huggingface dataset:
Postdivisional Synthesis of theSporosarcina ureaeDNA Translocase SpoIIIE either in the Mother Cell or in the Prespore EnablesBacillus subtilisTo Translocate DNA from the Mother Cell to the Prespore
Authors: Chary Vasant K. , Piggot Patrick J. 
Published: January 2003

ABSTRACTThe differentiation of vegetative cells ofBacillus subtilisinto spores involves asymmetric cell division, which precedes complete chromosome partitioning. The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division. We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation. We have expressed thespoIIIEhomologue fromSporosarcina ureaeinB. subtilisunder the control of different promoters. Expression from either a weak mother cell-specific (σE) promoter or a weak prespore-specific (σF) promoter partly complemented the sporulation defect of aspoIIIE36mutant; however, expression from a strong prespore-specific (σF) promoter did not. DNA translocation from the mother cell to the prespore was assayed usingspoIIQ-lacZinserted atthrC; transcription ofspoIIQoccurs only in the prespore. Translocation ofthrC::spoIIQ-lacZinto the prespore occurred efficiently whenspoIIIESuwas expressed from the weak σE- or σF-controlled promoters but not when it was expressed from the strong σF-controlled promoter. It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis.
Requirements for Cu
A
and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier
Pseudomonas putida
Authors: Wunsch Patrick , Herb Margitta , Wieland Hagen , Schiek Ulrike M. , Zumft Walter G. 
Published: January 2003

ABSTRACT

Bacterial nitrous oxide (N
2
O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N
2
O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu
A
site in the periplasm. There are seven putative maturation factors, encoded by
nosA
,
nosD
,
nosF
,
nosY
,
nosL
,
nosX
, and
sco
. We wanted to determine the indispensable proteins by expressing
nos
genes from
Pseudomonas stutzeri
in the nondenitrifying organism
Pseudomonas putida
. An in silico study of denitrifying bacteria revealed that
nosL
,
nosX
(or a homologous gene,
apbE
), and
sco
, but not
nosA
, coexist consistently with the N
2
O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with
nosRZ
in
trans
was sufficient to produce catalytically active holo-N
2
O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with
P. stutzeri
, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu
A
metallation. Both of these were found to be dispensable elements for N
2
O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N
2
O reductase maturation.
Plant Lectin-Like Bacteriocin from a Rhizosphere-Colonizing
Pseudomonas
Isolate
Authors: Parret Annabel H. A. , Schoofs Geert , Proost Paul , De Mot René 
Published: January 2003

ABSTRACT

Rhizosphere isolate
Pseudomonas
sp. strain BW11M1, which belongs to the
Pseudomonas putida
cluster, secretes a heat- and protease-sensitive bacteriocin which kills
P. putida
GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a Tn
Mod
mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into
Escherichia coli
cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in
E. coli
. This gene was designated
llp
A (
l
ectin-
l
ike
p
utidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the
llpA
gene also conveyed bacteriocin production to several
Pseudomonas fluorescens
strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described
Pseudomonas
bacteriocins.
Heme-Responsive Transcriptional Activation of
Bordetella bhu
Genes
Authors: Vanderpool Carin K. , Armstrong Sandra K. 
Published: January 2003

ABSTRACT

Bordetella pertussis
and
Bordetella bronchiseptica
, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the
bhuRSTUV
genes. Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions. The
hurIR
(heme uptake regulator) genes were previously identified upstream of the
bhuRSTUV
gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators. In this study,
B. pertussis
and
B. bronchiseptica
Δ
hurI
mutants, predicted to lack an ECF σ factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin. Genetic complementation of Δ
hurI
strains with plasmid-borne
hurI
restored wild-type levels of heme utilization.
B. bronchiseptica
Δ
hurI
mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor;
hurI
in
trans
restored heme-inducible BhuR expression to the mutant and resulted in BhuR overproduction. Transcriptional analyses with
bhuR-lacZ
fusion plasmids confirmed that
bhuR
transcription was activated in iron-starved cells in response to heme compounds. Heme-responsive
bhuR
transcription was not observed in mutant BRM23, indicating that
hurI
is required for positive regulation of
bhu
gene expression. Furthermore,
bhuR
was required for heme-inducible
bhu
gene activation, supporting the hypothesis that positive regulation of
bhuRSTUV
occurs by a surface signaling mechanism involving the heme-iron receptor BhuR.
Recombination Activity of a Distinctive Integron-Gene Cassette System Associated withPseudomonas stutzeriPopulations in Soil
Authors: Holmes Andrew J. , Holley Marita P. , Mahon Andrew , Nield Blair , Gillings Michael , Stokes H. W. 
Published: January 2003

ABSTRACTClass 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation ofPseudomonas stutzeristrain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar ofP. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. TheintIandattIcomponents of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (thePseudomonassubfamily). The recombination activity ofP. stutzeriintegron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognateattIsite and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of thePseudomonas59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present inP. stutzeripossess recombination activity and represent a hot spot for genomic diversity in this species.
In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake inStreptomyces coelicolor
Authors: Nothaft Harald , Parche Stephan , Kamionka Annette , Titgemeyer Fritz 
Published: January 2003

ABSTRACTHPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organismStreptomyces coelicolor, the encoding gene,ptsH, was deleted. TheptsHmutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparentKmof 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putativefruKAgene locus led to identification of the fructose-specific enzyme II permease encoded by thefruAgene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis ofptsHrevealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in aptsHbackground, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS inS. coelicolor.
Characterization of a Bifunctional Archaeal Acyl Coenzyme A Carboxylase
Authors: Chuakrut Songkran , Arai Hiroyuki , Ishii Masaharu , Igarashi Yasuo 
Published: January 2003

ABSTRACT

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from
Acidianus brierleyi
. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α
4
β
4
γ
4
subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent

K
m

for acetyl-CoA was 0.17 ± 0.03 mM, with a
V
max
of 43.3 ± 2.8 U mg
−1
, and the

K
m

for propionyl-CoA was 0.10 ± 0.008 mM, with a
V
max
of 40.8 ± 1.0 U mg
−1
. This result showed that
A. brierleyi
acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of
Bacteria
than to those of
Eucarya
. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.
A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects
Authors: Mori Hiroyuki , Akiyama Yoshinori , Ito Koreaki 
Published: January 2003

ABSTRACT

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the
secY39
defect. A specific
secE
mutation, designated
secE105
, was thus isolated. This mutation proved to be identical with the
prlG2
mutation and to suppress a number of cold-sensitive
secY
mutations. However, other
prlG
mutations did not effectively suppress the
secY
defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.
Characterization of the Stringent Response and

rel
Bbu

Expression in
Borrelia burgdorferi
Authors: Bugrysheva Julia , Dobrikova Elena Y. , Sartakova Marina L. , Caimano Melissa J. , Daniels Thomas J. , Radolf Justin D. , Godfrey Henry P. , Cabello Felipe C. 
Published: January 2003

ABSTRACT

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious
Borrelia burgdorferi
strain B31 and infectious
B. burgdorferi
strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in
B. burgdorferi
under these conditions. Here we report that (p)ppGpp levels in
B. burgdorferi
growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of
B. burgdorferi
(

rel
Bbu

) was produced by B31 as by N40 during in vitro growth (2 ± 0.5 and 4 ± 0.8 fg of

rel
Bbu

mRNA/ng of total
Borrelia
RNA, respectively). Although the amounts of N40

rel
Bbu

mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in

rel
Bbu

mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to
Bacillus subtilis
Rel
Bsu
were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in

rel
Bbu

promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40

rel
Bbu

ATG start codon and identical σ
70
-like promoters were identified by primer extension and sequencing analysis.

rel
Bbu

complemented an
Escherichia coli
CF1693
relA spoT
double mutant for growth on M9 minimal medium, and the transformed cells produced

rel
Bbu

mRNA. These results indicate that

rel
Bbu

is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in
B. burgdorferi
, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in
E. coli
and that (p)ppGpp may not be responsible for growth rate control.
Rates and Consequences of Recombination between rRNA Operons
Authors: Hashimoto Joel G. , Stevenson Bradley S. , Schmidt Thomas M. 
Published: January 2003

ABSTRACT

A mutant strain of
Escherichia coli
was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (
sacB-neo
) into the small-subunit rRNA-encoding gene
rrs
in the
rrnB
operon. During growth in a complex medium, the cassette was lost from the population, and a complete
rrs
gene was restored at a rate of 5 × 10
−9
per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored
rrnB
operons was 1 to 2% higher than that of the mutant strain. The
rrnB
operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in
rrnC
, -
E
, and -
G
. In 2 of the 14 strains in which
rrnB
was restored, the spacer region had the same length as the spacer region in
rrnA
, -
D
, and -
H
. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of
rrnB
suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in
E. coli
. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.
The Absence of FtsH Metalloprotease Activity Causes Overexpression of the σ
W
-Controlled
pbpE
Gene, Resulting in Filamentous Growth of
Bacillus subtilis
Authors: Zellmeier Stephan , Zuber Ulrich , Schumann Wolfgang , Wiegert Thomas 
Published: January 2003

ABSTRACT

FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins. For
Bacillus subtilis
, little is known about the target proteins of FtsH protease. Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth. Comparison of the intracellular proteomes of wild-type and
ftsH
knockout strains revealed that at least nine proteins accumulated in the absence of
ftsH
, four of which could be identified. Two of these proteins turned out to be members of the σ
W
regulon. Accumulation of one of these σ
W
-controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail. We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (
pbpE
) in
ftsH
null mutants. The filamentous growth phenotype of Δ
ftsH
strains was abolished in a Δ
ftsH
Δ
pbpE
double knockout. In
ftsH
wild-type strains with the
pbpE
gene under regulatable control,
pbpE
overexpression caused filamentation of the cells. DNA macroarray analysis revealed that most genes of the σ
W
regulon are transcribed at elevated levels in an
ftsH
mutant. The influence of FtsH on σ
W
-controlled genes is discussed.
Escherichia coliEndoribonucleases Involved in Cleavage of Bacteriophage T4 mRNAs
Authors: Otsuka Yuichi , Ueno Hiroyuki , Yonesaki Tetsuro 
Published: January 2003

ABSTRACTThedmdmutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying varioussocalleles, we could detect cleavages ofsocRNA in uninfected cells identical to those found indmdmutant-infected cells. We isolated fiveEscherichia colimutant strains in which thedmdmutant was able to grow. One of these strains completely suppressed thedmdmutant-specific cleavages ofsocRNA. The loci of theE. colimutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing thedmdmutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5′-truncatedsocRNA, a stable form in T4-infected cells regardless of the presence of admdmutation, is generated by RNase E.
Characterization of SrgA, a
Salmonella enterica
Serovar Typhimurium Virulence Plasmid-Encoded Paralogue of the Disulfide Oxidoreductase DsbA, Essential for Biogenesis of Plasmid-Encoded Fimbriae
Authors: Bouwman C. W. , Kohli M. , Killoran A. , Touchie G. A. , Kadner R. J. , Martin N. L. 
Published: January 2003

ABSTRACT

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of
Salmonella enterica
serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example,
Escherichia coli
, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family
Enterobacteriaceae
. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the
S. enterica
serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not.
pefA
and
srgA
were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in
pef
gene expression are also involved in
srgA
expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of
S. enterica
serovar Typhimurium.