bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacteriophage T4 Self-Assembly: In Vitro Reconstitution of Recombinant gp2 into Infectious Phage
Authors: Wang G. R. , Vianelli A. , Goldberg E. B. 
Published: July 2002

ABSTRACTT4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in therecBCD+host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2+phage infection (H+) with tails from a gp2+or gp2−phage infection (T+or T−), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H−) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV+cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2+phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.
Autoinduction of 2,4-Diacetylphloroglucinol Biosynthesis in the Biocontrol Agent
Pseudomonas fluorescens
CHA0 and Repression by the Bacterial Metabolites Salicylate and Pyoluteorin
Authors: Schnider-Keel Ursula , Seematter Arnaud , Maurhofer Monika , Blumer Caroline , Duffy Brion , Gigot-Bonnefoy Cécile , Reimmann Cornelia , Notz Regina , Défago Geneviève , Haas Dieter , Keel Christoph 
Published: July 2002

ABSTRACT

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of
Pseudomonas fluorescens
strain CHA0 to control plant diseases caused by soilborne pathogens. A 2,4-DAPG-negative Tn
5
insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn
5
insertion site was determined. Four open reading frames were identified, two of which were homologous to
phlA
, the first gene of the 2,4-DAPG biosynthetic operon, and to the
phlF
gene encoding a pathway-specific transcriptional repressor. The Tn
5
insertion was located in an open reading frame, tentatively named
phlH
, which is not related to known
phl
genes. In wild-type CHA0, 2,4-DAPG production paralleled expression of a
phlA′-′lacZ
translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing
phlA′-′lacZ
expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a
phlA
mutant, which was unable to produce 2,4-DAPG. In a
phlF
mutant, 2,4-DAPG production was enhanced severalfold and
phlA′-′lacZ
was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The
phlF
mutant was insensitive to 2,4-DAPG addition. A transcriptional
phlA-lacZ
fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of
phlA′-′lacZ
and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus
Fusarium
. In the
phlF
mutant, these compounds did not affect
phlA′-′lacZ
expression and 2,4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of
phlA′-′lacZ
expression was confirmed by using
Escherichia coli
as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on
phlF
function, may help
P. fluorescens
to produce homeostatically balanced amounts of extracellular metabolites.
Effect of Temperature on Stability and Activity of Elongation Factor 2 Proteins from Antarctic and Thermophilic Methanogens
Authors: Thomas Torsten , Cavicchioli Ricardo 
Published: July 2002

ABSTRACT

Despite the presence and abundance of archaea in low-temperature environments, little information is available regarding their physiological and biochemical properties. In order to investigate the adaptation of archaeal proteins to low temperatures, we purified and characterized the elongation factor 2 (EF-2) protein from the Antarctic methanogen
Methanococcoides burtonii
, which was expressed in
Escherichia coli
, and compared it to the recombinant EF-2 protein from a phylogenetically related thermophile,
Methanosarcina thermophila
. Using differential scanning calorimetry to assess protein stability and enzyme assays for the intrinsic GTPase activity, we identified biochemical and biophysical properties that are characteristic of the cold-adapted protein. This includes a higher activity at low temperatures caused by a decrease of the activation energy necessary for GTP hydrolysis and a decreased activation energy for the irreversible denaturation of the protein, which indicates a less thermostable structure. Comparison of the in vitro properties of the proteins with the temperature-dependent characteristics of growth of the organisms indicates that additional cytoplasmic factors are likely to be important for the complete thermal adaptation of the proteins in vivo. This is the first study to address thermal adaptation of proteins from a free-living, cold-adapted archaeon, and our results indicate that the ability of the Antarctic methanogen to adapt to the cold is likely to involve protein structural changes.
Multiple Factors Independently RegulatehilAand Invasion Gene Expression inSalmonella entericaSerovar Typhimurium
Authors: Lucas Robin L. , Lostroh C. Phoebe , DiRusso Concetta C. , Spector Michael P. , Wanner Barry L. , Lee Catherine A. 
Published: July 2002

ABSTRACTHilA activates the expression ofSalmonella entericaserovar Typhimurium invasion genes. To learn more about regulation ofhilA, we isolated Tn5mutants exhibiting reducedhilAand/or invasion gene expression. In addition to expected mutations, we identified Tn5insertions inpstS,fadD,flhD,flhC, andfliA. Analysis of thepstSmutant indicates thathilAand invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting thathilAexpression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of thefadDmutant indicates thathilAis regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulatehilAexpression.flhDCandfliAencode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies withflhCandfliAmutants indicate that FliZ, which is encoded in an operon withfliA, activates expression ofhilA, linking regulation ofhilAwith motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulatehilAexpression and invasion. In summary, our screen has identified several distinct pathways that can modulateS. entericaserovar Typhimurium's ability to expresshilAand invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.
Characterization of the Promoter Elements for the Staphylococcal Enterotoxin D Gene
Authors: Zhang Shuping , Stewart George C. 
Published: July 2002

ABSTRACT

Deletion analysis of the promoter for the
Staphylococcus aureus
enterotoxin D determinant indicated that a 52-bp sequence, from −34 to +18, was sufficient for
sed
promoter function and
agr
regulation. A consensus −10 Pribnow box sequence, a less conserved −35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity.
Genetic Variation and Evolutionary Origin of the Direct Repeat Locus of
Mycobacterium tuberculosis
Complex Bacteria
Authors: van Embden J. D. A. , van Gorkom T. , Kremer K. , Jansen R. , van der Zeijst B. A. M. , Schouls L. M. 
Published: July 2002

ABSTRACT

The direct repeat region in
Mycobacterium tuberculosis
complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of
M. tuberculosis
complex strains. We identified 94 different spacer sequences among 26
M. tuberculosis
complex strains. No sequence homology was found between any of these spacers and
M. tuberculosis
DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare
Mycobacterium canettii
taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.
Probing the CD Lumenal Loop Region of the D2 Protein of Photosystem II in
Synechocystis
sp. Strain PCC 6803 by Combinatorial Mutagenesis
Authors: Keilty Anna T. , Ermakova-Gerdes Svetlana Y. , Vermaas Wim F. J. 
Published: July 2002

ABSTRACT

The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in
Synechocystis
sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.
Effects of
bfp
Mutations on Biogenesis of Functional Enteropathogenic
Escherichia coli
Type IV Pili
Authors: Anantha Ravi P. , Stone Kelly D. , Donnenberg Michael S. 
Published: July 2002

ABSTRACT

Enteropathogenic
Escherichia coli
expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of
E. coli
. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the
bfp
cluster,
bfpG
,
bfpB
,
bfpC
,
bfpD
,
bfpP
, and
bfpH
, as well as the further analysis of a previously described
bfpA
mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in
bfpB
, which encodes an outer membrane protein;
bfpD
, which encodes a putative nucleotide-binding protein; and
bfpG
and
bfpC
, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in
bfpP
, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in
bfpH
, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many
bfp
gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.
Functional Analysis of the Galactosyltransferases Required for Biosynthesis of
d
-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of
Klebsiella pneumoniae
Authors: Guan Shukui , Clarke Anthony J. , Whitfield Chris 
Published: July 2002

ABSTRACT

d
-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-
d
-Gal
f
-(1→3)-α-
d
-Gal
p
-(1→], that is found in the lipopolysaccharide of
Klebsiella pneumoniae
O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of
d
-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of
d
-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from
Escherichia coli
K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The
d
-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-Glc
p
NAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into
d
-galactan I biosynthesis by the bifunctional
wbbO
gene product, which sequentially adds one Gal
p
and one Gal
f
residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Gal
f
precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of
d
-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Gal
p
residue to form a lipid-linked tetrasaccharide. Chain extension of the
d
-galactan I polymer requires WbbM for Gal
p
transferase, together with Gal
f
transferase activity provided by WbbO. Comparison of the biosynthetic pathways for
d
-galactan I and the polymannose
E. coli
O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.
Combined, Functional Genomic-Biochemical Approach to Intermediary Metabolism: Interaction of Acivicin, a Glutamine Amidotransferase Inhibitor, with
Escherichia coli
K-12
Authors: Smulski Dana R. , Huang Lixuan L. , McCluskey Michael P. , Reeve Mary Jane Gladnick , Vollmer Amy C. , Van Dyk Tina K. , LaRossa Robert A. 
Published: July 2002

ABSTRACT

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of
Escherichia coli
in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from
E. coli
, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process.
Multiple Mobile Promoter Regions for the Rare Carbapenem Resistance Gene of
Bacteroides fragilis
Authors: Podglajen I. , Breuil J. , Rohaut A. , Monsempes C. , Collatz E. 
Published: July 2002

ABSTRACT

Two novel insertion sequences (IS), IS
1187
and IS
1188
, are described upstream from the carbapenem resistance gene
cfiA
in strains of
Bacteroides fragilis
. Mapping, with the RACE procedure, of transcription start sites of
cfiA
in these and two other previously reported IS showed that transcription of this rarely encountered gene is initiated close to a variety of
B. fragilis
consensus promoter sequences, as recently defined (D. P. Bayley, E. R. Rocha, and C. J. Smith, FEMS Microbiol. Lett. 193:149–154, 2000). In the cases of IS
1186
and IS
1188
, these sequences overlap with putative Eς
70
promoter sequences, while in IS
942
and IS
1187
such sequences can be observed either upstream or downstream of the
B. fragilis
promoters.
Efg1, a Morphogenetic Regulator in
Candida albicans
, Is a Sequence-Specific DNA Binding Protein
Authors: Leng Ping , Lee Philip R. , Wu Hong , Brown Alistair J. P. 
Published: July 2002

ABSTRACT

Efg1 is essential for hyphal development in the human pathogen
Candida albicans
under most conditions. Efg1 is related to basic helix-loop-helix regulators, and therefore most workers presume that Efg1 is a transcription factor. Here we confirm that Efg1 is a DNA binding protein that can interact specifically with the E box.
Structural and Functional Characterization of IS
679
and IS
66
-Family Elements
Authors: Han Chang-Gyun , Shiga Yasuyuki , Tobe Toru , Sasakawa Chihiro , Ohtsubo Eiichi 
Published: July 2002

ABSTRACT

A new insertion sequence (IS) element, IS
679
(2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic
Escherichia coli
B171. IS
679
has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called
tnpA, tnpB
, and
tnpC
). A plasmid carrying a composite transposon (Tn
679
) with the kanamycin resistance gene flanked by an intact IS
679
sequence and an IS
679
fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS
679
. A transposition assay done with a mating system showed that Tn
679
could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn
679
duplicated an 8-bp sequence at the target site. Tn
679
derivatives with a deletion in each ORF of IS
679
did not transpose, finding indicative that all three IS
679
ORFs are essential for transposition. The
tnpA
and
tnpC
products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS
679
are present in
Agrobacterium, Escherichia, Rhizobium, Pseudomonas
, and
Vibrio
spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS
66
family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS
679
and, like IS
679
, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS
679
, but many have a mutation(s) in an ORF(s). In almost all of the elements,
tnpB
is located in the −1 frame relative to
tnpA
, such that the initiation codon of
tnpB
overlaps the TGA termination codon of
tnpA
. In contrast,
tnpC
, separated from
tnpB
by a space of ca. 20 bp, is located in any one of three frames relative to
tnpB
. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.
Genome Sequence and Comparative Analysis of the Solvent-Producing BacteriumClostridium acetobutylicum
Authors: Nölling Jörk , Breton Gary , Omelchenko Marina V. , Makarova Kira S. , Zeng Qiandong , Gibson Rene , Lee Hong Mei , Dubois JoAnn , Qiu Dayong , Hitti Joseph ,   GTC Sequencing Center Production Finishing, and Bioinformatics Teams, Wolf Yuri I. , Tatusov Roman L. , Sabathe Fabrice , Doucette-Stamm Lynn , Soucaille Philippe , Daly Michael J. , Bennett George N. , Koonin Eugene V. , Smith Douglas R. 
Published: July 2002

ABSTRACTThe genome sequence of the solvent-producing bacteriumClostridium acetobutylicumATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison ofC. acetobutylicumtoBacillus subtilisreveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, theC. acetobutylicumgenome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not withB. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome ofC. acetobutylicumcomprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified inB. subtilisare missing inC. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.