bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mannitol-1-Phosphate Dehydrogenase (MtlD) Is Required for Mannitol and Glucitol Assimilation in
Bacillus subtilis
: Possible Cooperation of
mtl
and
gut
Operons
Authors: Watanabe Shouji , Hamano Miyuki , Kakeshita Hiroshi , Bunai Keigo , Tojo Shigeo , Yamaguchi Hirotake , Fujita Yasutaro , Wong Sui-Lam , Yamane Kunio 
Published: August 2003

ABSTRACT

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in
Bacillus subtilis
. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the
mtl
operon, whereas glucitol induced both
mtl
and
gut
operons. The
B. subtilis mtl
operon consists of
mtlA
(encoding enzyme IICBA
mt1
) and
mtlD
, and its transcriptional regulator gene,
mtlR
, is located 14.4 kb downstream from the
mtl
operon on the chromosome. The
mtlA
,
mtlD
, and
mtlR
mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-β-
d
-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (P
spac
promoter) located in
mtlA
, and this site corresponds to the upstream region of
mtlD
. Insertion mutants of
mtlD
harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of
mtlA
could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the
mtl
operon but not to a DNA fragment containing the
gut
promoter region.
The Virulence Activator AphA Links Quorum Sensing to Pathogenesis and Physiology in
Vibrio cholerae
by Repressing the Expression of a Penicillin Amidase Gene on the Small Chromosome
Authors: Kovacikova Gabriela , Lin Wei , Skorupski Karen 
Published: August 2003

ABSTRACT

Activation of the
tcpPH
promoter on the
Vibrio
pathogenicity island by AphA and AphB initiates the
Vibrio cholerae
virulence cascade and is regulated by quorum sensing through the repressive action of HapR on
aphA
expression. To further understand how the chromosomally encoded AphA protein activates
tcpPH
expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation. This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities. Searching the
V. cholerae
genome for this binding site permitted the identification of a second one upstream of a penicillin V amidase (PVA) gene on the small chromosome. AphA binds to and footprints this site, which overlaps the
pva
transcriptional start, consistent with its role as a repressor at this promoter. Since
aphA
expression is under quorum-sensing control, the response regulators LuxO and HapR also influence
pva
expression. Thus,
pva
is repressed at low cell density when AphA levels are high, and it is derepressed at high cell density when AphA levels are reduced. Penicillin amidases are thought to function as scavengers for phenylacetylated compounds in the nonparasitic environment. That AphA oppositely regulates the expression of
pva
from that of virulence, together with the observation that PVA does not play a role in virulence, suggests that these activities are coordinated to serve
V. cholerae
in different biological niches.
Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis
Authors: Grimek T. L. , Holden H. , Rayment I. , Escalante-Semerena J. C. 
Published: August 2003

ABSTRACTTheprpBgene ofSalmonella entericaserovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ions; equimolar concentrations of Mn2+ions were a poor substitute for Mg2+(28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. TheKmof PrpB for 2-MIC was measured at 19 μM, with akcatof 105 s−1. Mutations in theprpBgene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpBK121A, PrpBH125A, and PrpBR122Kmutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases inkcatfor 2-MIC lyase activity, respectively. The PrpBD58Aand PrpBC123Aproteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg2+ion.
Allosteric Regulation of
Bacillus subtilis
NAD Kinase by Quinolinic Acid
Authors: Garavaglia Silvia , Galizzi Alessandro , Rizzi Menico 
Published: August 2003

ABSTRACT

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized
Bacillus subtilis
NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.
Autoinducer 2 Production byStreptococcusgordoniiDL1 and the Biofilm Phenotype of aluxSMutant Are Influenced by Nutritional Conditions
Authors: Blehert David S. , Palmer Robert J. , Xavier Joao B. , Almeida Jonas S. , Kolenbrander Paul E. 
Published: August 2003

ABSTRACTTheluxSgene, present in many bacterial genera, encodes the autoinducer 2 (AI-2) synthase. AI-2 has been implicated in bacterial signaling, and this study investigated its role in biofilm formation byStreptococcusgordonii, an organism that colonizes human tooth enamel within the first few hours after professional cleaning. Northern blotting and primer extension analyses revealed thatS.gordoniiluxSis monocistronic. AI-2 production was dependent on nutritional conditions, and maximum AI-2 induction was detected whenS.gordoniiwas grown in the presence of serum and carbonate. In planktonic cultures, AI-2 production rose sharply during the transition from exponential to stationary phase, and the AI-2 concentration peaked approximately 4 h into stationary phase. AnS.gordoniiluxSmutant that did not produce AI-2 was constructed by homologous recombination. Complementation of the mutant by insertion of an intactluxSgene into the chromosome in tandem with the disrupted gene restored AI-2 production to a level similar to that of the wild-type strain. In planktonic culture, no growth differences were observed between the mutant and wild-type strains when five different media were used. However, when grown for 4 h as biofilms in 25% human saliva under flow, theluxSmutant formed tall microcolonies that differed from those formed by the wild-type and complemented mutant strains. Biofilms of theluxSmutant exhibited finger-like projections of cells that extended into the flow cell lumen. Thus, the inability to produce AI-2 is associated with altered microcolony architecture withinS.gordoniibiofilms formed in saliva during a time frame consistent with initial colonization of freshly cleaned enamel surfaces.
Probing Conservation of HAMP Linker Structure and Signal Transduction Mechanism through Analysis of Hybrid Sensor Kinases
Authors: Appleman J. Alex , Chen Li-Ling , Stewart Valley 
Published: August 2003

ABSTRACTThe HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic α-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in theEscherichia colinitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.
A Novel IS Element, IS
621
, of the IS
110
/IS
492
Family Transposes to a Specific Site in Repetitive Extragenic Palindromic Sequences in
Escherichia coli
Authors: Choi Sunju , Ohta Shinya , Ohtsubo Eiichi 
Published: August 2003

ABSTRACT

An
Escherichia coli
strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS
621
) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS
621
was homologous to the
piv
genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS
621
as the query revealed that the protein also has partial homology to transposases encoded by the IS
110
/IS
492
family elements, which were known to have partial homology to PIV. This indicates that IS
621
belongs to the IS
110
/IS
492
family but is most closely related to the
piv
genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS
621
belongs to the
piv
gene group, which is distinct from the IS
110
/IS
492
family elements, which form several groups. PIV proteins and transposases encoded by the IS
110
/IS
492
family elements, including IS
621
, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS
621
was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS
621
may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS
110
/IS
492
family that also transpose to specific sites in the repeated sequences, as does IS
621
. IS
621
does not have terminal inverted repeats like most of the IS
110
/IS
492
family elements. The terminal sequences of IS
621
have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS
621
initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.
Benzoate-Coenzyme A Ligase from
Thauera aromatica
: an Enzyme Acting in Anaerobic and Aerobic Pathways
Authors: Schühle Karola , Gescher Johannes , Feil Ulrich , Paul Michael , Jahn Martina , Schägger Hermann , Fuchs Georg 
Published: August 2003

ABSTRACT

In the denitrifying member of the β-
Proteobacteria
Thauera aromatica
, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on
V
max
/

K
m

, the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.
Haemophilus influenzae
Rd Lacks a Stringently Conserved Fatty Acid Biosynthetic Enzyme and Thermal Control of Membrane Lipid Composition
Authors: Wang Haihong , Cronan John E. 
Published: August 2003

ABSTRACT

The organization of the fatty acid synthetic genes of
Haemophilus influenzae
Rd is remarkably similar to that of the paradigm organism,
Escherichia coli
K-12, except that no homologue of the
E. coli fabF
gene is present. This finding is unexpected, since
fabF
is very widely distributed among bacteria and is thought to be the generic 3-ketoacyl-acyl carrier protein (ACP) synthase active on long-chain-length substrates. However,
H. influenzae
Rd contains a homologue of the
E. coli fabB
gene, which encodes a 3-ketoacyl-ACP synthase required for unsaturated fatty acid synthesis, and it seemed possible that the
H. influenzae
FabB homologue might have acquired the functions of FabF.
E. coli
mutants lacking
fabF
function are unable to regulate the compositions of membrane phospholipids in response to growth temperature. We report in vivo evidence that the enzyme encoded by the
H. influenzae fabB
gene has properties essentially identical to those of
E. coli
FabB and lacks FabF activity. Therefore,
H. influenzae
grows without FabF function. Moreover, as predicted from studies of the
E. coli fabF
mutants,
H. influenzae
is unable to change the fatty acid compositions of its membrane phospholipids with growth temperature. We also demonstrate that the
fabB
gene of
Vibrio cholerae
El Tor N16961 does not contain a frameshift mutation as was previously reported.
Mapping the MinE Site Involved in Interaction with the MinD Division Site Selection Protein of
Escherichia coli
Authors: Ma Lu-Yan , King Glenn , Rothfield Lawrence 
Published: August 2003

ABSTRACT

Interactions between the MinD and MinE proteins are required for proper placement of the
Escherichia coli
division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed
minE
mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.
Phenotype MicroArray Analysis of
Escherichia coli
K-12 Mutants with Deletions of All Two-Component Systems
Authors: Zhou Lu , Lei Xiang-He , Bochner Barry R. , Wanner Barry L. 
Published: August 2003

ABSTRACT

Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in
Escherichia coli
K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including
creBC
-regulated genes (
cbrA
and
cbrBC
),
phoBR
-regulated genes (
phoA
,
phoH
,
phnCDEFGHIJKLMNOP
,
psiE
, and
ugpBAECQ
),
csgD
,
luxS
, and
rpoS
. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.
Genomic Profiling of Iron-Responsive Genes in
Salmonella enterica
Serovar Typhimurium by High-Throughput Screening of a Random Promoter Library
Authors: Bjarnason Jaime , Southward Carolyn M. , Surette Michael G. 
Published: August 2003

ABSTRACT

The importance of iron to bacteria is shown by the presence of numerous iron-scavenging and transport systems and by many genes whose expression is tightly regulated by iron availability. We have taken a global approach to gene expression analysis of
Salmonella enterica
serovar Typhimurium in response to iron by combining efficient, high-throughput methods with sensitive, luminescent reporting of gene expression using a random promoter library. Real-time expression profiles of the library were generated under low- and high-iron conditions to identify iron-regulated promoters, including a number of previously identified genes. Our results indicate that approximately 7% of the genome may be regulated directly or indirectly by iron. Further analysis of these clones using a Fur titration assay revealed three separate classes of genes; two of these classes consist of Fur-regulated genes. A third class was Fur independent and included both negatively and positively iron-responsive genes. These may reflect new iron-dependent regulons. Iron-responsive genes included iron transporters, iron storage and mobility proteins, iron-containing proteins (redox proteins, oxidoreductases, and cytochromes), transcriptional regulators, and the energy transducer
tonB
. By identifying a wide variety of iron-responsive genes, we extend our understanding of the global effect of iron availability on gene expression in the bacterial cell.
Identification of a Regulated Alkaline Phosphatase, a Cell Surface-Associated Lipoprotein, inMycobacterium smegmatis
Authors: Kriakov Jordan , Lee Sun hee , Jacobs William R. 
Published: August 2003

ABSTRACTAlthough alkaline phosphatases are common in a wide variety of bacteria, there has been no prior evidence for alkaline phosphatases inMycobacterium smegmatis. Here we report that transposon insertions in thepstoperon, encoding homologues of an inorganic phosphate transporter, leads to constitutive expression of a protein with alkaline phosphatase activity. DNA sequence analysis revealed thatM. smegmatisdoes indeed have aphoAgene that shows high homology to otherphoAgenes. TheM. smegmatis phoAgene was shown to be induced by phosphate starvation and thus negatively regulated by thepstoperon. Interestingly, the putativeM. smegmatisPhoA has a hydrophobic N-terminal domain which resembles a lipoprotein signal sequence. TheM. smegmatisPhoA was demonstrated to be an exported protein associated with the cell surface. Furthermore, immunoprecipitation of PhoA from [14C]acetate-labeledM. smegmatiscell lysates demonstrated that this phosphatase is a lipoprotein.
Identification of Long Intergenic Repeat Sequences Associated with DNA Methylation Sites in
Caulobacter crescentus
and Other α-Proteobacteria
Authors: Chen Swaine L. , Shapiro Lucy 
Published: August 2003

ABSTRACT

A systematic search for motifs associated with CcrM DNA methylation sites revealed four long (>100-bp) motifs (CIR sequences) present in up to 21 copies in
Caulobacter crescentus
. The CIR1 and CIR2 motifs exhibit a conserved inverted repeat organization, with a CcrM site in the center of one of the repeats.
Mutational Analysis of Cell Wall Biosynthesis inMycobacterium avium
Authors: Laurent Jean-Pierre , Hauge Kirsten , Burnside Kellie , Cangelosi Gerard 
Published: August 2003

ABSTRACTThe cell wall of the environmental pathogenMycobacterium aviumis important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, “transposome” insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of theM.aviumgenome.