bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization by Genetic Complementation of
aroB
-Encoded Dehydroquinate Synthase from
Mycobacterium tuberculosis
H37Rv and Its Heterologous Expression and Purification
Authors: de Mendonça Jordana Dutra , Ely Fernanda , Palma Mario Sergio , Frazzon Jeverson , Basso Luiz Augusto , Santos Diógenes Santiago 
Published: June 2007

ABSTRACT

The recent recrudescence of
Mycobacterium tuberculosis
infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in
M. tuberculosis
, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The
aroB
-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-
d
-
arabino
-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the
aroB
gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The
Escherichia coli
AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant
M. tuberculosis
dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with
M. tuberculosis
dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.
Distribution of Megaplasmids inLactobacillus salivariusand Other Lactobacilli
Authors: Li Yin , Canchaya Carlos , Fang Fang , Raftis Emma , Ryan Kieran A. , van Pijkeren Jan-Peter , van Sinderen Douwe , O'Toole Paul W. 
Published: June 2007

ABSTRACTThe genome ofLactobacillus salivariusUCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains ofL. salivariusisolated from humans and animals all harbor a megaplasmid, which hybridized with therepAandrepEreplication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118repAprobe were also found in some strains ofL. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of therepEandgroELsequences of 28L. salivariusstrains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids inL. salivariuswas highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six otherLactobacillusspecies from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids inL. salivarius, and restricted carriage by otherLactobacillusspecies, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology ofLactobacillus.
Cyclic AMP Directly Activates NasP, an
N
-Acyl Amino Acid Antibiotic Biosynthetic Enzyme Cloned from an Uncultured β-Proteobacterium
Authors: Clardy Jon , Brady Sean F. 
Published: June 2007

ABSTRACT

The cyclic AMP (cAMP)-dependent biosynthesis of
N
-acylphenylalanine antibiotics by NasP, an environmental DNA-derived
N
-acyl amino acid synthase, is controlled by an NasP-associated cyclic nucleotide-binding domain and is independent of the global cAMP signal transducer, cAMP receptor protein. A 16S rRNA gene sequence found on the same environmental DNA cosmid as NasP is most closely related to 16S sequences from β-proteobacteria.
New
Streptococcus pneumoniae
Clones in Deceased Wild Chimpanzees
Authors: Chi Fang , Leider Michaela , Leendertz Fabian , Bergmann Carina , Boesch Christophe , Schenk Svenja , Pauli Georg , Ellerbrok Heinz , Hakenbeck Regine 
Published: June 2007

ABSTRACT

In wild chimpanzees in the Taï National Park, Côte d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of
Streptococcus pneumoniae
were identified in deceased apes on the basis of multilocus sequence typing analysis and
ply
,
lytA
, and
pbp2x
sequences. The findings suggest that virulent
S. pneumoniae
occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans.
Bacteriochlorophyllide
c
C-8
2
and C-12
1
Methyltransferases Are Essential for Adaptation to Low Light in
Chlorobaculum tepidum
Authors: Chew Aline Gomez Maqueo , Frigaard Niels-Ulrik , Bryant Donald A. 
Published: June 2007

ABSTRACT

Bacteriochlorophyll (BChl)
c
is the major photosynthetic pigment in the green sulfur bacterium
Chlorobaculum tepidum
, in which it forms protein-independent aggregates that function in light harvesting. BChls
c
,
d
, and
e
are found only in chlorosome-producing bacteria and are unique among chlorophylls because of methylations that occur at the C-8
2
and C-12
1
carbons. Two genes required for these methylation reactions were identified and designated
bchQ
(CT1777) and
bchR
(CT1320). BchQ and BchR are members of the radical
S
-adenosylmethionine (SAM) protein superfamily; each has sequence motifs to ligate a [4Fe-4S] cluster, and we propose that they catalyze the methyl group transfers.
bchQ
,
bchR
, and
bchQ bchR
mutants of
C. tepidum
were constructed and characterized. The
bchQ
mutant produced BChl
c
that was not methylated at C-8
2
, the
bchR
mutant produced BChl
c
that was not methylated at C-12
1
, and the double mutant produced [8-ethyl, 12-methyl]-BChl
c
that lacked methylation at both the C-8
2
and C-12
1
positions. Compared to the wild type, the Q
y
absorption bands for BChl
c
in the mutant cells were narrower and blue shifted to various extents. All three mutants grew slower and had a lower cellular BChl
c
content than the wild type, an effect that was especially pronounced at low light intensities. These observations show that the C-8
2
and C-12
1
methylations of BChl
c
play important roles in the adaptation of
C. tepidum
to low light intensity. The data additionally suggest that these methylations also directly or indirectly affect the regulation of the BChl
c
biosynthetic pathway.
Genetic Structure of the
nadA
and
nadB
Antivirulence Loci in
Shigella
spp
Authors: Prunier Anne-Laure , Schuch Raymond , Fernández Reinaldo E. , Maurelli Anthony T. 
Published: June 2007

ABSTRACT

Comparison of
nadA
and
nadB
in 14
Shigella
strains and enteroinvasive
Escherichia coli
versus
E. coli
showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of
Shigella
organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.
A Novel Exclusion Mechanism for Carbon Resource Partitioning in
Aggregatibacter actinomycetemcomitans
Authors: Brown Stacie A. , Whiteley Marvin 
Published: June 2007

ABSTRACT

The bacterium
Aggregatibacter actinomycetemcomitans
is a common commensal of the human oral cavity and the putative causative agent of the disease localized aggressive periodontitis.
A. actinomycetemcomitans
is a slow-growing bacterium that possesses limited metabolic machinery for carbon utilization. This likely impacts its ability to colonize the oral cavity, where growth and community composition is mediated by carbon availability. We present evidence that in the presence of the in vivo relevant carbon substrates glucose, fructose, and lactate
A. actinomycetemcomitans
preferentially metabolizes lactate. This preference for lactate exists despite the fact that
A. actinomycetemcomitans
grows faster and obtains higher cell yields during growth with carbohydrates. The preference for lactate is mediated by a novel exclusion mechanism in which metabolism of lactate inhibits carbohydrate uptake. Coculture studies reveal that
A. actinomycetemcomitans
utilizes lactate produced by the oral bacterium
Streptococcus gordonii
, suggesting the potential for cross-feeding in the oral cavity.
SadC Reciprocally Influences Biofilm Formation and Swarming Motility via Modulation of Exopolysaccharide Production and Flagellar Function
Authors: Merritt Judith H. , Brothers Kimberly M. , Kuchma Sherry L. , O'Toole George A. 
Published: June 2007

ABSTRACTPseudomonas aeruginosahas served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of thesadCgene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ΔsadCmutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that thesadCgene is part of a genetic pathway that allows for the concomitant regulation of these aspects ofP. aeruginosasurface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation asP. aeruginosatransitions from a planktonic to a surface-associated lifestyle.
BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility byPseudomonas aeruginosaPA14
Authors: Kuchma Sherry L. , Brothers Kimberly M. , Merritt Judith H. , Liberati Nicole T. , Ausubel Frederick M. , O'Toole George A. 
Published: June 2007

ABSTRACTThe intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors inPseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this genebifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifAmutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by thepellocus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifAmutant but does not contribute to the observed swarming defect. The ΔbifAmutation also results in decreased flagellar reversals. Based on epistasis studies with the previously describedsadBgene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.
EbpR Is Important for Biofilm Formation by Activating Expression of the Endocarditis and Biofilm-Associated Pilus Operon (
ebpABC
) of
Enterococcus faecalis
OG1RF
Authors: Bourgogne Agathe , Singh Kavindra V. , Fox Kristina A. , Pflughoeft Kathryn J. , Murray Barbara E. , Garsin Danielle A. 
Published: June 2007

ABSTRACT

We identify ef1090 (renamed
ebpR
) and show its importance for the transcriptional regulation of expression of the
Enterococcus faecalis
pilus operon,
ebpABC
. An
ebpR
deletion (Δ
ebpR
) mutant was found to have reduced
ebpABC
expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.
The Antirepressor Needed for Induction of Linear Plasmid-Prophage N15 Belongs to the SOS Regulon
Authors: Mardanov Andrey V. , Ravin Nikolai V. 
Published: June 2007

ABSTRACTThe physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene,antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter ofantC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.
Expression ofhurP, a Gene Encoding a Prospective Site 2 Protease, Is Essential for Heme-Dependent Induction ofbhuRinBordetella bronchiseptica
Authors: King-Lyons Natalie D. , Smith Kelsy F. , Connell Terry D. 
Published: June 2007

ABSTRACTExpression of thehurIR bhuRSTUVheme utilization locus inBordetella bronchisepticais coordinately controlled by the global iron-dependent regulator Fur and the extracytoplasmic function sigma factor HurI. Activation of HurI requires transduction of a heme-dependent signal via HurI, HurR, and BhuR, a three-component heme-dependent regulatory system. In silico searches of theB. bronchisepticagenome to identify other genes that encode additional participants in this heme-dependent regulatory cascade revealedhurP, an open reading frame encoding a polypeptide with homology to (i) RseP, a site 2 protease (S2P) ofEscherichia colirequired for modifying the cytoplasmic membrane protein RseA, and (ii) YaeL, an S2P ofVibrio choleraerequired for modification of the cytoplasmic membrane protein TcpP. A mutant ofB. bronchisepticadefective forhurPwas incapable of regulating expression of BhuR in a heme-dependent manner. Furthermore, thehurPmutant was unable to utilize hemin as a sole source of nutrient Fe. These defects in hemin utilization and heme-dependent induction of BhuR were restored when recombinanthurP(or recombinantrseP) was introduced into the mutant. Introduction ofhurPinto ayaeLmutant ofV. choleraealso complemented its S2P defect. These data provided strong evidence that protease activity and cleavage site recognition was conserved in HurP, RseP, and YaeL. The data are consistent with a model in which HurP functionally modifies HurR, a sigma factor regulator that is essential for heme-dependent induction ofbhuR.
Dual Role of OhrR as a Repressor and an Activator in Response to Organic Hydroperoxides inStreptomyces coelicolor
Authors: Oh So-Young , Shin Jung-Ho , Roe Jung-Hye 
Published: June 2007

ABSTRACTOrganic hydroperoxide resistance in bacteria is achieved primarily through reducing oxidized membrane lipids. The soil-inhabiting aerobic bacteriumStreptomyces coelicolorcontains three paralogous genes for organic hydroperoxide resistance:ohrA,ohrB, andohrC. TheohrAgene is transcribed divergently fromohrR, which encodes a putative regulator of MarR family. Both theohrAandohrRgenes were induced highly by various organic hydroperoxides. TheohrAgene was induced through removal of repression by OhrR, whereas theohrRgene was induced through activation by OhrR. Reduced OhrR bound to theohrA-ohrRintergenic region, which contains a central (primary) and two adjacent (secondary) inverted-repeat motifs that overlap with promoter elements. Organic peroxide decreased the binding affinity of OhrR for the primary site, with a concomitant decrease in cooperative binding to the adjacent secondary sites. The single cysteine C28 in OhrR was involved in sensing oxidants, as determined by substitution mutagenesis. The C28S mutant of OhrR bound to the intergenic region without any change in binding affinity in response to organic peroxides. These results lead us to propose a model for the dual action of OhrR as a repressor and an activator inS. coelicolor. Under reduced conditions, OhrR binds cooperatively to the intergenic region, repressing transcription from both genes. Upon oxidation, the binding affinity of OhrR decreases, with a concomitant loss of cooperative binding, which allows RNA polymerase to bind to both theohrAandohrRpromoters. The loosely bound oxidized OhrR can further activate transcription from theohrRpromoter.
Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication
Authors: Contursi Patrizia , Cannio Raffaele , Prato Santina , She Qunxin , Rossi Mosè , Bartolucci Simonetta 
Published: June 2007

ABSTRACTpSSVx fromSulfolobus islandicusstrain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle ofS. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5′ end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.
Natural Merodiploidy of thelux-ribOperon ofPhotobacterium leiognathifrom Coastal Waters of Honshu, Japan
Authors: Ast Jennifer C. , Urbanczyk Henryk , Dunlap Paul V. 
Published: June 2007

ABSTRACTSequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis usingluxgenes, however, is based on the assumptions that theluxgenes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains ofPhotobacterium leiognathicarry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes,luxCDABEG-ribEBHA. Merodiploidlux-ribstrains ofP. leiognathiwere detected during sequence analysis ofluxA. To define the gene content, organization, and sequence of eachlux-riboperon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain,lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis oflnuch.13.1 defined two complete, physically separate, and apparently functional operons, designatedlux-rib1andlux-rib2.P. leiognathistrainslelon.2.1 andlnuch.21.1 were also found to carrylux-rib1andlux-rib2, whereas ATCC 25521Tapparently carries onlylux-rib1. Inlnuch.13.1,lelon.2.1,lnuch.21.1, and ATCC 25521T,lux-rib1is flanked upstream bylumQandputAand downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flanklux-rib2oflnuch.13.1, and the chromosomal location oflux-rib2apparently differs inlnuch.13.1,lelon.2.1, andlnuch.21.1. Phylogenetic analysis demonstrated thatlux-rib1andlux-rib2are more closely related to each other than either one is to theluxandribgenes of other bacterial species, which rules out interspecies lateral gene transfer as the origin oflux-rib2inP. leiognathi;lux-rib2apparently arose within a previously unsampled or extinctP. leiognathilineage. Analysis of 170 additional strains ofP. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a singlelux-riboperon and 68 that carry multiplelux-riboperons. Strains bearing a singlelux-riboperon were obtained throughout the geographic sampling range, whereaslux-ribmerodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy ofluxorribgenes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.
Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium
Rhodobacter capsulatus
Authors: Tremblay Pier-Luc , Drepper Thomas , Masepohl Bernd , Hallenbeck Patrick C. 
Published: June 2007

ABSTRACT

Both
Rhodobacter capsulatus
PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the
R. capsulatus
membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a
glnK
mutant, more GlnB was found to be membrane associated under these conditions. In a
glnB
mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in
R. capsulatus
.
Sortase C-Mediated Anchoring of BasI to the Cell Wall Envelope of
Bacillus anthracis
Authors: Marraffini Luciano A. , Schneewind Olaf 
Published: June 2007

ABSTRACT

Vegetative forms of
Bacillus anthracis
replicate in tissues of an infected host and precipitate lethal anthrax disease. Upon host death, bacilli form dormant spores that contaminate the environment, thereby gaining entry into new hosts where spores germinate and once again replicate as vegetative forms. We show here that sortase C, an enzyme that is required for the formation of infectious spores, anchors BasI polypeptide to the envelope of predivisional sporulating bacilli. BasI anchoring to the cell wall requires the active site cysteine of sortase C and an LPNTA motif sorting signal at the C-terminal end of the BasI precursor. The LPNTA motif of BasI is cleaved between the threonine (T) and the alanine (A) residue; the C-terminal carboxyl group of threonine is subsequently amide linked to the side chain amino group of diaminopimelic acid within the wall peptides of
B. anthracis
peptidoglycan.