bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conversion of Methionine to Cysteine in
Bacillus subtilis
and Its Regulation
Authors: Hullo Marie-Françoise , Auger Sandrine , Soutourina Olga , Barzu Octavian , Yvon Mireille , Danchin Antoine , Martin-Verstraete Isabelle 
Published: December 2006

ABSTRACT

Bacillus subtilis
can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in
Escherichia coli
were tested. Both CysK and YrhA have an
O
-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine β-synthase activity using
O
-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine γ-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for
cysK
or
yrhB
grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an Δ
yrhA
mutant or a
luxS
mutant, inactivated for the
S
-ribosyl-homocysteinase step of the
S
-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a Δ
yrhA ΔcysK
or
cysE
mutant did not grow at all under the same conditions. The
yrhB
and
yrhA
genes form an operon together with
yrrT
,
mtnN
, and
yrhC
. The expression of the
yrrT
operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the
yrrT
promoter region in gel-shift experiments. The addition of
O
-acetyl-serine prevented the formation of this protein-DNA complex.
Complete Sequence Analysis of Novel Plasmids from Emetic and Periodontal
Bacillus cereus
Isolates Reveals a Common Evolutionary History among the
B. cereus
-Group Plasmids, Including
Bacillus anthracis
pXO1
Authors: Rasko David A. , Rosovitz M. J. , Økstad Ole Andreas , Fouts Derrick E. , Jiang Lingxia , Cer Regina Z. , Kolstø Anne-Brit , Gill Steven R. , Ravel Jacques 
Published: December 2006

ABSTRACT

The plasmids of the members of the
Bacillus cereus
sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example,
Bacillus anthracis
contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines
Bacillus thuringiensis
isolates. In this study the plasmids from
B. cereus
isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ∼272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ∼270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these
B. cereus
plasmids revealed a high degree of sequence similarity to the
B. anthracis
pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified
B. cereus
plasmids. Screening of a population of
B. cereus
group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic
B. cereus
isolates in the same way that pXO1 and pXO2 define the
B. anthracis
species.
The Timing ofcotEExpression AffectsBacillus subtilisSpore Coat Morphology but Not Lysozyme Resistance
Authors: Costa Teresa , Serrano Mónica , Steil Leif , Völker Uwe , Moran Charles P. , Henriques Adriano O. 
Published: December 2006

ABSTRACTThe synthesis of structural components and morphogenetic factors required for the assembly of theBacillus subtilisspore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σEfactor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. ThecotEgene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme.cotEis expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σKcontrol. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplantedcotEto progressively later classes of mother cell gene expression. This created an early class of mutants in whichcotEis expressed prior to engulfment completion and a late class in which expression ofcotEfollows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.
High-Affinity Binding of the Staphylococcal HarA Protein to Haptoglobin and Hemoglobin Involves a Domain with an Antiparallel Eight-Stranded β-Barrel Fold
Authors: Dryla Agnieszka , Hoffmann Bernd , Gelbmann Dieter , Giefing Carmen , Hanner Markus , Meinke Andreas , Anderson Annaliesa S. , Koppensteiner Walter , Konrat Robert , von Gabain Alexander , Nagy Eszter 
Published: December 2006

ABSTRACT

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for “near transporter”) present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione
S
-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (

K
D

) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel β-barrel with the strand order (-β1
↓
-β2
↑
-β3
↓
-β6
↑
-β5
↓
-β4
↑
-β7
↓
-β8
↑
), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.
Mutations in the
Yersinia pseudotuberculosis
Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells
Authors: Davis Alison J. , Mecsas Joan 
Published: December 2006

ABSTRACT

The trafficking of effectors, termed Yops, from
Yersinia
spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD)
yscF
mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two
yscF
TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I
yscF
TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II
yscF
TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.
The
l
-Tartrate/Succinate Antiporter TtdT (YgjE) of
l
-Tartrate Fermentation in
Escherichia coli
Authors: Kim Ok Bin , Unden Gottfried 
Published: December 2006

ABSTRACT

Escherichia coli
ferments
l
-tartrate under anaerobic conditions in the presence of an additional electron donor to succinate. The carrier for
l
-tartrate uptake and succinate export and its relation to the general C
4
-dicarboxylate carriers DcuA, DcuB, and DcuC were studied. The secondary carrier TtdT, encoded by the
ttdT
(previously called
ygjE
) gene, is required for the uptake of
l
-tartrate. The
ttdT
gene is located downstream of the
ttdA
and
ttdB
genes, encoding the
l
-tartrate dehydratase TtdAB. Analysis of mRNA by reverse transcription-PCR showed that
ttdA
,
ttdB
, and
ttdT
are cotranscribed. Deletion of
ttdT
abolished growth by
l
-tartrate and degradation of
l
-tartrate completely. Bacteria containing TtdT catalyze
l
-tartrate or succinate uptake and specific heterologous
l
-tartrate/succinate antiporting.
d
-Tartrate is not a substrate for TtdT. TtdT operates preferentially in the direction of tartrate uptake and succinate excretion. The Dcu carriers do not support anaerobic growth on
l
-tartrate or
l
-tartrate transport. TtdT is related in sequence and function to CitT, which catalyzes heterologous citrate/succinate antiporting in citrate fermentation.
Rare Group I Intron with Insertion Sequence Element in a Bacterial Ribonucleotide Reductase Gene
Authors: Meng Qing , Zhang Yi , Liu Xiang-Qin 
Published: December 2006

ABSTRACT
A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.
CapA, an Autotransporter Protein ofCampylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut
Authors: Ashgar Sami S. A. , Oldfield Neil J. , Wooldridge Karl G. , Jones Michael A. , Irving Greg J. , Turner David P. J. , Ala'Aldeen Dlawer A. A. 
Published: December 2006

ABSTRACTTwo putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence ofCampylobacter jejuniNCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. AcapAinsertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization byCampylobacter. In view of this demonstrated role, we propose that CapA stands forCampylobacteradhesion protein A.
Genetic Analysis of a Novel Pathway for
d
-Xylose Metabolism in
Caulobacter crescentus
Authors: Stephens Craig , Christen Beat , Fuchs Thomas , Sundaram Vidyodhaya , Watanabe Kelly , Jenal Urs 
Published: December 2006

ABSTRACT

Genetic data suggest that the oligotrophic freshwater bacterium
Caulobacter crescentus
metabolizes
d
-xylose through a pathway yielding α-ketoglutarate, comparable to the recently described
l
-arabinose degradation pathway of
Azospirillum brasilense
. Enzymes of the
C. crescentus
pathway, including an NAD
+
-dependent xylose dehydrogenase, are encoded in the xylose-inducible
xylXABCD
operon (CC0823-CC0819).
Evolution of
Mycobacterium ulcerans
and Other Mycolactone-Producing Mycobacteria from a Common
Mycobacterium marinum
Progenitor
Authors: Yip Marcus J. , Porter Jessica L. , Fyfe Janet A. M. , Lavender Caroline J. , Portaels Françoise , Rhodes Martha , Kator Howard , Colorni Angelo , Jenkin Grant A. , Stinear Tim 
Published: December 2006

ABSTRACT

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with
Mycobacterium ulcerans
, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the
M. ulcerans
pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both
M. ulcerans
and
Mycobacterium marinum
. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common
M. marinum
progenitor to form a genetically cohesive group among a more diverse assemblage of
M. marinum
strains. Like
M. ulcerans
, the fish and frog MPM show multiple copies of the insertion sequence IS
2404
. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.
Bacillus subtilisGene Cluster Involved in Calcium Carbonate Biomineralization
Authors: Barabesi Chiara , Galizzi Alessandro , Mastromei Giorgio , Rossi Mila , Tamburini Elena , Perito Brunella 
Published: December 2006

ABSTRACTCalcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation byBacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the EuropeanB. subtilisFunctional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA,ysiA,ysiB,etfB, andetfA) called thelcfAoperon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutatedlcfAoperon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-β-d-thiogalactopyranoside) control, the last gene,etfA, was identified as essential for the precipitation process. To verify cotranscription in thelcfAoperon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.
Linker Regions of the RhaS and RhaR Proteins
Authors: Kolin Ana , Jevtic Visnja , Swint-Kruse Liskin , Egan Susan M. 
Published: December 2006

ABSTRACT
Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.
“Cold-Sensitive” Mutants of the Lac Repressor
Authors: Barker Andrew , Oehler Stefan , Müller-Hill Benno 
Published: December 2006

ABSTRACT
Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.
Glucose Uptake Pathway-Specific Regulation of Synthesis of Neotrehalosadiamine, a Novel Autoinducer Produced in
Bacillus subtilis
Authors: Inaoka Takashi , Ochi Kozo 
Published: December 2006

ABSTRACT

Neotrehalosadiamine (3,3′-diamino-3,3′-dideoxy-α,β-trehalose; NTD) is an amino-sugar antibiotic produced by several
Bacillus
species that functions as an autoinducer by activating its own biosynthetic operon,
ntdABC
. We previously reported that the introduction of a certain
rpoB
mutation (
rpoB5
) into
Bacillus subtilis
enables the cells to overproduce NTD.
B. subtilis
mini-Tn
10
transposant libraries have been screened for genes that affect NTD production. Inactivation of
ccpA
, which encodes a major transcriptional regulator of carbon catabolite regulation, markedly reduced NTD production. By contrast, inactivation of
glcP
, which is situated just downstream of
ntdABC
and encodes a glucose/mannose:H
+
symport permease, stimulated NTD production. Overexpression of
glcP
led to the repression of
ntdABC
expression (and thus NTD production) in response to GlcP-mediated glucose uptake. These results suggest that CcpA-mediated catabolite activation of
ntdABC
expression occurs in response to the increase of the in vivo concentration of fructose-1,6-bisphosphate via glucose-6-phosphate and that GlcP-mediated glucose repression of
ntdABC
expression occurs in association with the increase of the in vivo concentration of unphosphorylated glucose. In addition, Northern analysis showed that
glcP
is transcribed from the
ntdABC
promoter through transcription readthrough at the
ntdABC
transcription terminator site, which enables NTD to function as a modulator of glucose uptake through the stimulation of
ntdABC-glcP
transcription, even in wild-type (
rpoB
+
) cells. A trace amount (0.5 to 3 μg/ml) of NTD was sufficient to ensure expression of
glcP
, thus demonstrating the physiological role of “antibiotic” in the producing bacteria by functioning as an autoinducer for glucose uptake modulation.
Genome Analyses of Three Strains of
Rhodobacter sphaeroides
: Evidence of Rapid Evolution of Chromosome II
Authors: Choudhary M. , Zanhua Xie , Fu Y. X. , Kaplan S. 
Published: December 2006

ABSTRACT

Three strains of
Rhodobacter sphaeroides
of diverse origin have been under investigation in our laboratory for their genome complexities, including the presence of multiple chromosomes and the distribution of essential genes within their genomes. The genome of
R. sphaeroides
2.4.1 has been completely sequenced and fully annotated, and now two additional strains (ATCC 17019 and ATCC 17025) of
R. sphaeroides
have been sequenced. Thus, genome comparisons have become a useful approach in determining the evolutionary relationships among different strains of
R. sphaeroides
. In this study, the concatenated chromosomal sequences from the three strains of
R. sphaeroides
were aligned, using Mauve, to examine the extent of shared DNA regions and the degree of relatedness among their chromosome-specific DNA sequences. In addition, the exact intra- and interchromosomal DNA duplications were analyzed using Mummer. Genome analyses employing these two independent approaches revealed that strain ATCC 17025 diverged considerably from the other two strains, 2.4.1 and ATCC 17029, and that the two latter strains are more closely related to one another. Results further demonstrated that chromosome II (CII)-specific DNA sequences of
R. sphaeroides
have rapidly evolved, while CI-specific DNA sequences have remained highly conserved. Aside from the size variation of CII of
R. sphaeroides
, variation in sequence lengths of the CII-shared DNA regions and their high sequence divergence among strains of
R. sphaeroides
suggest the involvement of CII in the evolution of strain-specific genomic rearrangements, perhaps requiring strains to adapt in specialized niches.
Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 fromSulfolobus islandicusREN1H1 and Regulation of Its Replication Operon
Authors: Berkner Silvia , Lipps Georg 
Published: December 2006

ABSTRACTThe plasmid pRN1 fromSulfolobus islandicusREN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3′-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of theorf56-orf904cotranscript in vivo.