bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Phenotypic Changes in
Pseudomonas putida
in Response to Surface-Associated Growth
Authors: Sauer Karin , Camper Anne K. 
Published: July 2002

ABSTRACT

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte
Pseudomonas putida
following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of
P
.
putida
. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from
P
.
putida
and pure C
12
-HSL were added to 6-h planktonic cultures of
P
.
putida
, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that
P
.
putida
undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.
Colicin A Immunity Protein Interacts with the Hydrophobic Helical Hairpin of the Colicin A Channel Domain in the
Escherichia coli
Inner Membrane
Authors: Nardi Angèle , Corda Yves , Baty Daniel , Duché Denis 
Published: July 2002

ABSTRACT

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the
Escherichia coli
inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA α-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic α-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.
Transcriptional Regulation of
furA
and
katG
upon Oxidative Stress in
Mycobacterium smegmatis
Authors: Milano Anna , Forti Francesca , Sala Claudia , Riccardi Giovanna , Ghisotti Daniela 
Published: July 2002

ABSTRACT

The DNA region upstream of
katG
in
Mycobacterium smegmatis
was cloned and sequenced. The
furA
gene, highly homologous to
Mycobacterium tuberculosis furA
, mapped in this region. The
furA-katG
organization appears to be conserved among several mycobacteria. The transcription pattern of
furA
and
katG
in
M
.
smegmatis
upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both
furA
and
katG
was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5′ ends were identified for
furA
and
katG
, respectively. By cloning
M
.
smegmatis
and
M
.
tuberculosis
DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters,
pfurA
, located immediately upstream of the
furA
gene, and
pkatG
, located within the terminal part of the
furA
coding sequence. Transcription from
pfurA
was induced upon oxidative stress. A 23-bp sequence overlapping the
pfurA
−35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling
pfurA
activity. Transcription from a cloned
pkatG
, lacking the upstream
pfurA
region, was not induced upon oxidative stress, suggesting a
cis
-acting regulatory role of this region.
Analysis of the
pmsCEAB
Gene Cluster Involved in Biosynthesis of Salicylic Acid and the Siderophore Pseudomonine in the Biocontrol Strain
Pseudomonas fluorescens
WCS374
Authors: Mercado-Blanco Jesús , van der Drift Koen M. G. M. , Olsson Per E. , Thomas-Oates Jane E. , van Loon Leendert C. , Bakker Peter A. H. M. 
Published: July 2002

ABSTRACT

Mutants of
Pseudomonas fluorescens
WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb
Eco
RI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of
P. putida
. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (
pmsC
and
pmsB
) showed homologies with chorismate-utilizing enzymes; a third ORF (
pmsE
) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (
pmsA
). A putative promoter region and two predicted iron boxes were localized upstream of
pmsC
. We determined by reverse transcriptase-mediated PCR that the
pmsCEAB
genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in
P. putida
and
Escherichia coli
cells. In
E. coli
, deletions affecting the first ORF (
pmsC
) diminished SA production, whereas deletion of
pmsB
abolished it completely. The
pmsB
gene induced low levels of SA production in
E. coli
when expressed under control of the
lacZ
promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn
5
mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.
d
-Alanylation of Lipoteichoic Acid: Role of the
d
-Alanyl Carrier Protein in Acylation
Authors: Kiriukhin Michael Y. , Neuhaus Francis C. 
Published: July 2002

ABSTRACT

The
d
-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the
d
-alanine–
d
-alanyl carrier protein ligase (AMP) (Dcl) and the
d
-alanyl carrier protein (Dcp). The
dlt
operon encoding these proteins (
dltA
and
dltC
) also includes
dltB
and
dltD. dltB
encodes a putative transport system, while
dltD
encodes a protein which facilitates the binding of Dcp and Dcl for ligation with
d
-alanine and has thioesterase activity for mischarged
d
-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that
d
-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681–690, 1994). However, all efforts to identify an enzyme which catalyzes this
d
-alanylation process were unsuccessful. It was discovered that incubation of
d
-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this
d
-alanyl thioester.
d
-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated
d
-alanyl LTA, a time and concentration-dependent formation of
d
-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of
d
-alanyl-Dcp and stimulated the hydrolysis of
d
-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the
d
-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the
d
-alanyl ester residues between LTA and wall teichoic acid.
Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different
Authors: Seffernick Jennifer L. , de Souza Mervyn L. , Sadowsky Michael J. , Wackett Lawrence P. 
Published: July 2002

ABSTRACT

The gene encoding melamine deaminase (TriA) from
Pseudomonas
sp. strain NRRL B-12227 was identified, cloned into
Escherichia coli
, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from
Pseudomonas
sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.
Cytoplasmic RNA Polymerase in
Escherichia coli
Authors: Shepherd N. , Dennis P. , Bremer H. 
Published: July 2002

ABSTRACT

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase β and β′ subunits in DNA-free minicells from the minicell-producing
Escherichia coli
strain χ925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase β and β′ subunits in minicells produced by this strain corresponded to about 17% (or 2.5 μM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.
TraG-Like Proteins of DNA Transfer Systems and of theHelicobacter pyloriType IV Secretion System: Inner Membrane Gate for Exported Substrates?
Authors: Schröder Gunnar , Krause Sabine , Zechner Ellen L. , Traxler Beth , Yeo Hye-Jeong , Lurz Rudi , Waksman Gabriel , Lanka Erich 
Published: July 2002

ABSTRACTTraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such asHelicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.
Salt-Dependent Expression of Glucosylglycerol-Phosphate Synthase, Involved in Osmolyte Synthesis in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Marin Kay , Huckauf Jana , Fulda Sabine , Hagemann Martin 
Published: July 2002

ABSTRACT

The cyanobacterium
Synechocystis
sp. strain PCC 6803 is able to acclimate to levels of salinity ranging from freshwater to twice the seawater concentrations of salt by accumulating the compatible solute glucosylglycerol (GG). Expression of the
ggpS
gene coding for the key enzyme (glucosylglycerol-phosphate synthase) in GG synthesis was examined in detail. Under control conditions, the GgpS protein is stable, so that weak constitutive transcription of the
ggpS
gene resulted in a significant protein content. However, the enzyme activity was biochemically switched off, and no GG was detectable. After a salt shock, an immediate increase in mRNA content proportional to the salt content occurred, while the GgpS protein and GG contents rose in a linear manner. Furthermore, the stability of the
ggpS
mRNA increased transiently. In salt-acclimated cells expression of the
ggpS
gene, the GgpS protein content, and the amount of accumulated GG depended linearly on the external salt concentration. Mapping of the 5′ end of the
ggpS
transcript revealed a long nontranslated 5′ sequence and a putative typical cyanobacterial promoter, which did not show any obvious salt-regulatory element. The alternative σ factor σ
F
was found to be involved in salt-dependent regulation of
ggpS
, since in a σ
F
mutant induction of this gene was strongly reduced. The present study demonstrated that in addition to biochemical regulation of GgpS activity, alterations of
ggpS
expression are involved in regulation of GG synthesis in
Synechocystis
sp. strain PCC 6803. A model showing the interaction of the two regulatory levels is presented.
FpvA Receptor Involvement in Pyoverdine Biosynthesis in
Pseudomonas aeruginosa
Authors: Shen Jiangsheng , Meldrum Allison , Poole Keith 
Published: July 2002

ABSTRACT

Alignment of the
Pseudomonas aeruginosa
ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of
P. putida
. Deletion of
fpvA
from the chromosome of
P. aeruginosa
reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g.,
pvdD
) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type
fpvA
restored
pvd
expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of
fpvA
encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in
P. aeruginosa
.
Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain
Authors: Adler Julia , Bibi Eitan 
Published: July 2002

ABSTRACT

The hydrophobicity profile and sequence alignment of the
Escherichia coli
multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.
Genetic Variation at the O-Antigen Biosynthetic Locus in
Pseudomonas aeruginosa
Authors: Raymond Christopher K. , Sims Elizabeth H. , Kas Arnold , Spencer David H. , Kutyavin Tanya V. , Ivey Richard G. , Zhou Yang , Kaul Rajinder , Clendenning James B. , Olson Maynard V. 
Published: July 2002

ABSTRACT

The outer carbohydrate layer, or O antigen, of
Pseudomonas aeruginosa
varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the
P. aeruginosa
genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.
epr
Is Transcribed from a ς
D
Promoter and Is Involved in Swarming of
Bacillus subtilis
Authors: Dixit Madhulika , Murudkar Charuta S. , Rao K. Krishnamurthy 
Published: July 2002

ABSTRACT

Epr is a minor extracellular protease secreted by
Bacillus subtilis
168. In this study, we show that
epr
is transcribed by Eς
D
, the RNA polymerase associated with transcription of genes involved in chemotaxis and motility. Disruption of
epr
abolished swarming of
Bacillus subtilis
, suggesting its involvement in motility.
Characterization of the
parB
-Like
yyaA
Gene of
Bacillus subtilis
Authors: Sievers Jörg , Raether Brian , Perego Marta , Errington Jeff 
Published: July 2002

ABSTRACT

We have characterized the
yyaA
gene of
Bacillus subtilis
, located near the origin of chromosome replication (
oriC
). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the
yyaA
gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of
yyaA
caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.
In Vitro Identification of Rns-Regulated Genes
Authors: Munson George P. , Holcomb Lisa G. , Alexander Heather L. , Scott June R. 
Published: July 2002

ABSTRACTTo identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenicEscherichia coligenomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame ofyiiS, which lies immediately downstream of one MBP-Rns-bound fragment.
A Two-Component Regulator Mediates Population-Density-Dependent Expression of theBradyrhizobium japonicumNodulation Genes
Authors: Loh John , Lohar Dasharath P. , Andersen Brett , Stacey Gary 
Published: July 2002

ABSTRACTBradyrhizobium japonicum nodgene expression was previously shown to be population density dependent. Induction of thenodgenes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found inB. japonicumconditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded bynwsB,is required for the full expression of theB. japonicum nodYABCoperon. In addition, NwsB is also required for the population-density-dependent expression of bothnolAandnodD2. Expression ofnolAandnodD2in thenwsBmutant remained at a basal level, even at high culture densities. ThenwsBdefect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repressnodgene expression, the expression of anodY-lacZfusion in thenwsBmutant was unaffected by culture density. In plant assays withGUSfusions, nodules infected with the wild type showed nonodY-GUSexpression. In contrast,nodY-GUSexpression was not repressed in nodules infected with thenwsBmutant. Nodule competition assays between the wild type and thenwsBmutant revealed that the addition of conditioned medium resulted in a competitive advantage for thenwsBmutant.