bluuebunny/crossref_metadata_2025_split huggingface dataset:
Escherichia coliRNA Polymerase Recognition of a σ70-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif
Authors: Hook-Barnard India , Johnson Xanthia B. , Hinton Deborah M. 
Published: December 2006

ABSTRACTEscherichia coliσ70-dependent promoters have typically been characterized as either −10/−35 promoters, which have good matches to both the canonical −10 and the −35 sequences or as extended −10 promoters (TGn/−10 promoters), which have the TGn motif and an excellent match to the −10 consensus sequence. We report here an investigation of a promoter, Pminor, that has a nearly perfect match to the −35 sequence and has the TGn motif. However, Pminorcontains an extremely poor σ70−10 element. We demonstrate that Pminoris active both in vivo and in vitro and that mutations in either the −35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the −10 element more canonical, thus converting the −35/TGn promoter to a −35/−10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with Pminor, the DNA is single stranded from positions −11 to +4. We also demonstrate that transcription from Pminorincorporates nontemplated ribonucleoside triphosphates at the 5′ end of the Pminortranscript, which results in an anomalous assignment for the start site when primer extension analysis is used. Pminorrepresents one of the few −35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized −10/−35 and extended −10 promoters used byE. coliRNA polymerase.
Identification of a Gene Negatively Affecting Antibiotic Production and Morphological Differentiation inStreptomyces coelicolorA3(2)
Authors: Li Wencheng , Ying Xin , Guo Yuzheng , Yu Zhen , Zhou Xiufen , Deng Zixin , Kieser Helen , Chater Keith F. , Tao Meifeng 
Published: December 2006

ABSTRACTSC7A1 is a cosmid with an insert of chromosomal DNA fromStreptomyces coelicolorA3(2). Its insertion into the chromosome ofS. coelicolorstrains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frameSCO5582asnsdA, a genenegatively affectingStreptomycesdifferentiation. The disruption of chromosomalnsdAcaused the overproduction of spores and of three of four knownS. coelicolorantibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying thatnsdAin the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster.nsdAexpression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, aStreptomycesprotein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in someStreptomycesgenomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of thensdA-like geneSCO4114had no obvious phenotypic effects onS. coelicolor. ThensdAorthologueSAV2652inS. avermitiliscould complement theS. coelicolor nsdA-null mutant phenotype.
Identification of a
nisI
Promoter within the
nisABCTIP
Operon That May Enable Establishment of Nisin Immunity Prior to Induction of the Operon via Signal Transduction
Authors: Li Haiping , O'Sullivan Daniel J. 
Published: December 2006

ABSTRACT

Certain strains of
Lactococcus lactis
produce the broad-spectrum bacteriocin nisin, which belongs to the lantibiotic class of antimicrobial peptides. The genes encoding nisin are organized in three contiguous operons:
nisABTCIP
, encoding production and immunity (
nisI
);
nisRK
, encoding regulation; and
nisFEG
, also involved in immunity. Transcription of
nisABTCIP
and
nisFEG
requires autoinduction by external nisin via signal transducing by NisRK. This organization poses the intriguing question of how sufficient immunity (NisI) can be expressed when the nisin cluster enters a new cell, before it encounters external nisin. In this study, Northern analysis in both
Lactococcus
and
Enterococcus
backgrounds revealed that
nisI
mRNA was present under conditions when no
nisA
transcription was occurring, suggesting an internal promoter within the operon. The
nisA
transcript was significantly more stable than
nisI
, further substantiating this. Reverse transcriptase PCR analysis revealed that the transcription initiated just upstream from
nisI
. Fusing this region to a
lacZ
gene in a promoter probe vector demonstrated that a promoter was present. The transcription start site (TSS) of the
nisI
promoter was mapped at bp 123 upstream of the
nisI
translation start codon. Ordered 5′ deletions revealed that transcription activation depended on sequences located up to bp −234 from the TSS. The presence of poly(A) tracts and computerized predictions for this region suggested that a high degree of curvature may be required for transcription initiation. The existence of this
nisI
promoter is likely an evolutionary adaptation of the nisin gene cluster to enable its successful establishment in other cells following horizontal transfer.
Expression of the
pyr
Operon of
Lactobacillus plantarum
Is Regulated by Inorganic Carbon Availability through a Second Regulator, PyrR
2
, Homologous to the Pyrimidine-Dependent Regulator PyrR
1
Authors: Arsène-Ploetze Florence , Kugler Valérie , Martinussen Jan , Bringel Françoise 
Published: December 2006

ABSTRACT

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of
Lactobacillus plantarum
. At low IC levels, one-third of natural isolated
L. plantarum
strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO
2
-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the
pyrR
1
BCAa
1
Ab
1
DFE
operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated
pyr
genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the
L. plantarum
genome. PyrR
1
is an RNA-binding protein that regulates the
pyr
genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR
2
was investigated by allelic gene replacement. Unlike the
pyrR
1
mutant, the
ΔpyrR
2
strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO
2
was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in
pyrR
1
and
pyrR
2
mutants suggested that only PyrR
2
positively regulates the expression levels of the
pyr
genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR
1
and PyrR
2
in the
pyr
regulon expression.
Malonyl-Coenzyme A Reductase in the Modified 3-Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Archaeal
Metallosphaera
and
Sulfolobus
spp
Authors: Alber Birgit , Olinger Marc , Rieder Annika , Kockelkorn Daniel , Jobst Björn , Hügler Michael , Fuchs Georg 
Published: December 2006

ABSTRACT

Autotrophic members of the
Sulfolobales
(
Crenarchaeota
) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO
2
fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO
2
into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of
Metallosphaera sedula
contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum
Sulfolobus tokodaii
. The
Sulfolobus
gene was cloned and heterologously expressed in
Escherichia coli
, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H
+
→ malonate-semialdehyde + CoA + NADP
+
. In its native state it is associated with small RNA. Its activity was stimulated by Mg
2+
and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (

K
m

= 25 μM) and malonyl-CoA (

K
m

= 40 μM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from
Chloroflexus aurantiacus.
This shows that the autotrophic pathway in
Chloroflexus
and
Sulfolobaceae
has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.
Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides
Authors: Townsend Guy E. , Forsberg Lennart S. , Keating David H. 
Published: December 2006

ABSTRACTLeguminous plants and bacteria from the familyRhizobiaceaeform a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms betweenSinorhizobium melilotiand alfalfa requires biosynthesis of Nod factor, a β-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification.S. melilotialso produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants ofS. melilotiwith reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of theS. meliloticarbohydrate sulfotransferase, LpsS, inMesorhizobium loti. M. lotiparticipates in a determinate symbiosis with the legumeLotus japonicus. We showed thatM. lotiproduces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides inM. loti, we identified and disabled anM. lotihomolog of the sulfate-activating genes,nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled anM. loti cysHhomolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that thenodPQmutant, but not thecysHmutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume,Lotus japonicus. Interestingly, thenodPQmutant also exhibited a more rapid growth rate and appeared to outcompete wild-typeM. lotifor nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization inM. loti.
Lysophosphatidylethanolamine Is a Substrate for the Short-Chain Alcohol Dehydrogenase SocA from
Myxococcus xanthus
Authors: Avadhani Madhavi , Geyer Roland , White David C. , Shimkets Lawrence J. 
Published: December 2006

ABSTRACT

Short-chain alcohol dehydrogenases (SCADHs) synthesize a variety of intercellular signals and other chemically diverse products. It is difficult to predict the substrate of a SCADH on the basis of amino acid sequence homology, as the substrates are not known for most SCADHs. In
Myxococcus xanthus
, the SCADH CsgA is responsible for C signaling during fruiting body development, although the mechanism is unclear. Overexpression of the SCADH SocA compensates for the lack of CsgA and restores development and C signaling in
csgA
mutants. The potential of SocA in generating the C signal enzymatically was explored by developing a dehydrogenase assay-based screen to purify the SocA substrate(s). A SocA substrate was extracted from
M. xanthus
cells with acidified ethyl acetate and sequentially purified by solid-phase extraction on silica gel and by reverse-phase high-performance liquid chromatography. The fraction with the highest SocA dehydrogenase activity contained the lysophospholipid 1-acyl 2-hydroxy-
sn
-glycerophosphoethanolamine (lyso-PE) as indicated by the fragment ions and a phosphatidylethanolamine-specific neutral loss scan following liquid chromatography coupled to mass spectrometry. The abundant lysophospholipid with the mass
m/z
450 (molecular ion [M-H]
−
) had a monounsaturated acyl chain with 16 carbons. SocA oxidizes lyso-PE containing either saturated or unsaturated fatty acids but exhibits poor activity on
l
-α-glycerophosphorylethanolamine, suggesting that an acyl chain is important for activity. Of the five different head groups, only ethanolamine showed appreciable activity. The apparent

K
m

and
V
max
for lyso-PE 18:1 were 116 μM and 875 μmol min
−1
mg
−1
, respectively. The catalytic efficiency (
k
cat
/

K
m

) was 1 × 10
8
M
−1
s
−1
. The proposed product, 1-acyloxy-3-(2-aminoethylphosphatyl) acetone was unstable, and the fragmented products were unable to rescue
csgA
mutant development. The active fraction from thin-layer chromatography also contained an unidentified SocA substrate that had morphogenic properties.
Iron Starvation Triggers the Stringent Response and Induces Amino Acid Biosynthesis for Bacillibactin Production in
Bacillus subtilis
Authors: Miethke Marcus , Westers Helga , Blom Evert-Jan , Kuipers Oscar P. , Marahiel Mohamed A. 
Published: December 2006

ABSTRACT

Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved
Bacillus subtilis
cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.
Characterization of a Highly Conserved Island in the Otherwise Divergent
Bordetella holmesii
and
Bordetella pertussis
Genomes
Authors: Diavatopoulos D. A. , Cummings C. A. , van der Heide H. G. J. , van Gent M. , Liew S. , Relman D. A. , Mooi F. R. 
Published: December 2006

ABSTRACT

The recently discovered pathogen
Bordetella holmesii
has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of
B. holmesii
are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions,
B. holmesii
strains were analyzed by comparative genomic hybridization (CGH) to a
Bordetella pertussis
microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between
B. pertussis
and
B. holmesii
. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS
481
elements that may have been laterally transferred from
B. pertussis
to
B. holmesii
. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by
B. holmesii
may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between
B. pertussis
and
B. holmesii
may also explain the unusually high sequence identity of their 16S rRNA genes.
N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant
Authors: Kolkenbrock Stephan , Parschat Katja , Beermann Bernd , Hinz Hans-Jürgen , Fetzner Susanne 
Published: December 2006

ABSTRACTN-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation byArthrobacter nitroguajacolicusRü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (kcat/Km= 208 mM−1s−1), while among the aryl-acetylamides,o-carboxy- oro-nitro-substituted analogues were preferred overp-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams,N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1%N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes inKms of the enzyme and increasedkcats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasingkcatfor the formyl analogueN-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.
Functional Analysis of AtlA, the MajorN-Acetylglucosaminidase ofEnterococcus faecalis
Authors: Eckert Catherine , Lecerf Maxime , Dubost Lionel , Arthur Michel , Mesnage Stéphane 
Published: December 2006

ABSTRACTThe major peptidoglycan hydrolase ofEnterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is anN-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of theatlAgene inEscherichia colior partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms ofE. faecalisextracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from anatlAnull mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA inE. faecalisextracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.
Mycobacterium tuberculosisSigM Positively Regulates Esx Secreted Protein and Nonribosomal Peptide Synthetase Genes and Down Regulates Virulence-Associated Surface Lipid Synthesis
Authors: Raman Sahadevan , Puyang Xiaoling , Cheng Tan-Yun , Young David C. , Moody D. Branch , Husson Robert N. 
Published: December 2006

ABSTRACTTheMycobacterium tuberculosisgenome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigatedM. tuberculosisSigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in asigMmutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and thekasA-kasBoperon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in asigM-disrupted strain and is undetectable in asigMoverexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.
Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in
Escherichia coli
Authors: Correia Frederick F. , D'Onofrio Anthony , Rejtar Tomas , Li Lingyun , Karger Barry L. , Makarova Kira , Koonin Eugene V. , Lewis Kim 
Published: December 2006

ABSTRACT

Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in
Escherichia coli
. HipA is a “toxin” that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the
hipBA
operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg
2+
-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation.