bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Expression of the
Erwinia carotovora
subsp.
carotovora
Gene Encoding the Low-Molecular-Weight Bacteriocin Carocin S1
Authors: Chuang Duen-yau , Chien Yung-chei , Wu Huang-Pin 
Published: December 2006

ABSTRACT

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of
Erwinia carotovora
. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn
5
insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of
Erwinia carotovora
subsp.
carotovora
89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn
5
insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes,
caroS1K
(ORF2) and
caroS1I
(ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in
E. carotovora
subsp.
carotovora
Ea1068 and replicated in TH22-10 but could not be expressed in
Escherichia coli
(JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the
E. coli
cyclic AMP receptor protein binding site (−312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in
Pseudomonas aeruginosa
. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.
Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from
Streptococcus mitis
Authors: Jefferies Johanna , Nieminen Leena , Kirkham Lea-Ann , Johnston Calum , Smith Andrew , Mitchell Tim J. 
Published: December 2006

ABSTRACT

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of
Streptococcus mitis
isolates. We have sequenced the mitilysin gene from seven isolates of
S. mitis
. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions.
S. mitis
appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of
S. mitis
may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between
S. mitis
and
Streptococcus pneumoniae
, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.
Kinetic Analysis of the Assembly of the Outer Membrane Protein LamB in
Escherichia coli
Mutants Each Lacking a Secretion or Targeting Factor in a Different Cellular Compartment
Authors: Ureta Alejandro R. , Endres Robert G. , Wingreen Ned S. , Silhavy Thomas J. 
Published: December 2006

ABSTRACT

Outer membrane β-barrel proteins in gram-negative bacteria, such as
Escherichia coli
, must be translocated from their site of synthesis in the cytoplasm to the periplasm and finally delivered to the outer membrane. At least a dozen proteins located in the cytoplasm, the periplasm, and both the inner and outer membranes are required to catalyze this complex assembly process. At normal growth temperatures and conditions the transport and assembly processes are so fast that assembly intermediates cannot be detected. Using cells grown at a low temperature to slow the assembly process and pulse-chase analysis with immunodetection methods, we followed newly synthesized LamB molecules during their transit through the cell envelope. The quality and reproducibility of the data allowed us to calculate rate constants for three different subassembly reactions. This kinetic analysis revealed that
secB
and
secD
mutants exhibit nearly identical defects in precursor translocation from the cytoplasm. However, subsequent subassembly reaction rates provided no clear evidence for an additional role for SecD in LamB assembly. Moreover, we found that
surA
mutants are qualitatively indistinguishable from
yfgL
mutants, suggesting that the products of both of these genes share a common function in the assembly process, most likely the delivery of LamB to the YaeT assembly complex in the outer membrane.
Cys303 in the Histidine Kinase PhoR Is Crucial for the Phosphotransfer Reaction in the PhoPR Two-Component System in
Bacillus subtilis
Authors: Eldakak Amr , Hulett F. Marion 
Published: December 2006

ABSTRACT

The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the
resA
promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR
C303A
, exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR
C303A
and DTT are different. PhoR and PhoR
C303A
were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoR∼P, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoR∼P or PhoR
C303A
∼P and PhoP occurred rapidly; most label from PhoR∼P was transferred to PhoP, but only 10% of the label from PhoR
C303A
∼P was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoP∼P or PhoR autophosphatase activity was observed between PhoR and PhoR
C303A
that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR
C303
in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoR∼P to PhoP, which is stabilization that is required for efficient production of PhoP∼P.
CC1, a Novel Crenarchaeal DNA Binding Protein
Authors: Luo Xiao , Schwarz-Linek Uli , Botting Catherine H. , Hensel Reinhard , Siebers Bettina , White Malcolm F. 
Published: December 2006

ABSTRACT

The genomes of the related crenarchaea
Pyrobaculum aerophilum
and
Thermoproteus tenax
lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of
T. tenax
. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the
P. aerophilum
and
Aeropyrum pernix
genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in
T. tenax
. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.
Biosynthesis of Phosphoserine in the
Methanococcales
Authors: Helgadóttir Sunna , Rosas-Sandoval Guillermina , Söll Dieter , Graham David E. 
Published: December 2006

ABSTRACT

Methanococcus maripaludis
and
Methanocaldococcus jannaschii
produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA
Cys
with
l
-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in
Methanococcales
, cell extracts of
M. maripaludis
were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from
M. maripaludis
had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from
M. jannaschii
converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The
M. maripaludis
homolog complemented a
serC
mutation in the
Escherichia coli
phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.
Characterization ofHelicobacter pyloriLytic Transglycosylases Slt and MltD
Authors: Chaput Catherine , Labigne Agnès , Boneca Ivo G. 
Published: December 2006

ABSTRACTPeptidoglycan (PG) is a cell wall heteropolymer that is essential for cell integrity. PG hydrolases participate in correct assembly of the PG layer and have been shown to be required for cell division, cell daughter separation, and maintenance of bacterial morphology. In silico analysis of theHelicobacter pylorigenome resulted in identification of three potential hydrolases, Slt, MltD, and AmiA. This study was aimed at determining the roles of the putative lytic transglycosylases, Slt and MltD, inH. pylorimorphology, growth, and PG metabolism. Strain 26695 single mutants were constructed using a nonpolar kanamycin cassette. ThesltandmltDmutants formed normal bacillary and coccoid bacteria in the exponential and stationary phases, respectively. ThesltandmltDmutants had growth rates comparable to the growth rate of the parental strain. However, themltDmutant exhibited enhanced survival in the stationary phase compared to the wild type or thesltmutant. PG was purified from exponentially growing bacteria and from bacteria in the stationary phase, and its muropeptide composition was analyzed by high-pressure liquid chromatography. This analysis revealed changes in the muropeptide composition indicating that MltD and Slt have lytic transglycosylase activities. Glycan strand analysis suggested that Slt and MltD have exo and endo types of lytic transglycosylase activity, indicating that Slt is involved mainly in PG turnover and MltD is involved mainly in rearrangement of the PG layer. In this study, we determined the distinct roles of the lytic transglycosylases Slt and MltD in PG metabolism.
Mutational Analysis of the Flagellar Protein FliG: Sites of Interaction with FliM and Implications for Organization of the Switch Complex
Authors: Brown Perry N. , Terrazas Moises , Paul Koushik , Blair David F. 
Published: December 2006

ABSTRACT

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In
Escherichia coli
and
Salmonella
, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an α-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ
125-128
and R
160
(the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione
S
-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.
Global Gene Expression and Phenotypic Analysis of a
Vibrio cholerae rpoH
Deletion Mutant
Authors: Slamti Leyla , Livny Jonathan , Waldor Matthew K. 
Published: December 2006

ABSTRACT

Vibrio cholerae
, the cause of cholera, can grow in a variety of environments outside of human hosts. During infection, this pathogen must adapt to significant environmental alterations, including the elevated temperature of the human gastrointestinal tract. σ
32
, an alternative sigma factor encoded by
rpoH
, activates transcription of genes involved in the heat shock response in several bacterial species. Here, we assessed the role of σ
32
in
V. cholerae
physiology. In aggregate, our findings suggest that σ
32
promotes
V. cholerae
growth at temperatures ranging at least from 15°C to 42°C. Growth of the
rpoH
mutant was severely attenuated within the suckling mouse intestine, suggesting that σ
32
-regulated genes are critical for
V. cholerae
adaptation to conditions within the gastrointestinal tract. We defined the
V. cholerae
RpoH regulon by comparing the whole-genome transcription profiles of the wild-type and
rpoH
mutant strains after a temperature up-shift. Most of the
V. cholerae
genes expressed in an RpoH-dependent manner after heat shock encode proteins that influence protein fate, such as proteases and chaperones, or are of unknown function. Bioinformatic analyses of the microarray data were used to define a putative σ
32
consensus binding sequence and subsequently to identify genes that are likely to be directly regulated by RpoH in the whole
V. cholerae
genome.
Complete and Integrated Pyrene Degradation Pathway inMycobacterium vanbaaleniiPYR-1 Based on Systems Biology
Authors: Kim Seong-Jae , Kweon Ohgew , Jones Richard C. , Freeman James P. , Edmondson Ricky D. , Cerniglia Carl E. 
Published: December 2006

ABSTRACTMycobacterium vanbaaleniiPYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway forM. vanbaaleniiPYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with theM. vanbaaleniiPYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates througho-phthalate and the β-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism inM. vanbaaleniiPYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.
Phosphotransfer Reactions of the CbbRRS Three-Protein Two- Component System from
Rhodopseudomonas palustris
CGA010 Appear To Be Controlled by an Internal Molecular Switch on the Sensor Kinase
Authors: Romagnoli Simona , Tabita F. Robert 
Published: December 2006

ABSTRACT

The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the
cbb
I
CO
2
fixation operon of
Rhodopseudomonas palustris
, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSR
T189
and CbbSR
R96
(CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.
Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in
Escherichia coli
Authors: Brock Jay E. , Paz Robert L. , Cottle Patrick , Janssen Gary R. 
Published: December 2006

ABSTRACT

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many
Escherichia coli
genes, and addition of downstream adenine-rich sequences increases expression from several genes in
E. coli
. Here we describe site-directed mutagenesis of the
E. coli aroL
,
pncB
, and
cysJ
coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from
aroL-
,
pncB-
, and
cysJ-lacZ
fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from
aroL
- and
cysJ
-
lacZ
fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of
E. coli
translation.
Coordinate Expression of the Acetyl Coenzyme A Carboxylase Genes,
accB
and
accC
, Is Necessary for Normal Regulation of Biotin Synthesis in
Escherichia coli
Authors: Abdel-Hamid Ahmed M. , Cronan John E. 
Published: December 2006

ABSTRACT

Transcription of the biotin (
bio
) biosynthetic operon of
Escherichia coli
is negatively regulated by the BirA protein, an atypical repressor protein in that it is also an enzyme. The BirA-catalyzed reaction involves the covalent attachment of biotin to AccB, a subunit of acetyl coenzyme (acetyl-CoA) carboxylase. The two functions of BirA allow regulation of the
bio
operon to respond to the intracellular concentrations of both biotin and unbiotinylated AccB. We report here that
bio
operon expression is down-regulated by overproduction of AccC, another acetyl-CoA carboxylase subunit known to form a complex with AccB. This down-regulation is eliminated when AccB and AccC are coordinately overexpressed, but only when the AccB partner is competent to bind AccC. Under AccC overexpression conditions AccB is underbiotinylated. These findings can be explained by a model in which excess AccC sequesters AccB in a complex that is a poor substrate for biotinylation. The observed disruption of biotin synthesis and attachment provides an excellent rationale for the observation that in the vast majority of sequenced bacterial genomes AccB and AccC are encoded in a two-gene operon.
Heme Concentration Dependence and Metalloporphyrin Inhibition of the System I and II CytochromecAssembly Pathways
Authors: Richard-Fogal Cynthia L. , Frawley Elaine R. , Feissner Robert E. , Kranz Robert G. 
Published: December 2006

ABSTRACTStudies have indicated that specific heme delivery to apocytochromecis a critical feature of the cytochromecbiogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromesc, we engineeredEscherichia coliso that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (ΔhemA). The engineeredE. colistrains that produced recombinant system I (fromE. coli) or system II (fromHelicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromescwith other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II).