bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rhamnolipid Surfactant Production Affects Biofilm Architecture in
Pseudomonas aeruginosa
PAO1
Authors: Davey Mary E. , Caiazza Nicky C. , O'Toole George A. 
Published: January 2003

ABSTRACT

In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen
Pseudomonas aeruginosa
in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.
pIII
CTX
, a Predicted CTXφ Minor Coat Protein, Can Expand the Host Range of Coliphage fd To Include
Vibrio cholerae
Authors: Heilpern Andrew J. , Waldor Matthew K. 
Published: January 2003

ABSTRACT

CTXφ is a filamentous bacteriophage that encodes cholera toxin. CTXφ infection of its host bacterium,
Vibrio cholerae
, requires the toxin-coregulated pilus (TCP) and the products of the
V. cholerae tolQRA
genes. Here, we have explored the role of OrfU, a predicted CTXφ minor coat protein, in CTXφ infection. Prior to the discovery that it was part of a prophage,
orfU
was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXφ genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXφ infection as well as playing a role in CTXφ assembly and release. Deletion of
orfU
from CTXφ dramatically reduced the number of CTXφ virions detected in supernatants from CTXφ-bearing cells. This defect was complemented by expression of
orfU
in
trans
, thereby confirming a role for this gene in CTXφ assembly and/or release. To evaluate the requirement for OrfU in CTXφ infection, we introduced fragments of
orfU
into
gIII
in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect
V. cholerae
, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect
V. cholerae
in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXφ pIII, we propose to rename OrfU as pIII
CTX
. Our data also provide new evidence for a conserved pathway for filamentous phage infection.
The VirB4 Family of Proposed Traffic Nucleoside Triphosphatases: Common Motifs in Plasmid RP4 TrbE Are Essential for Conjugation and Phage Adsorption
Authors: Rabel Christian , Grahn A. Marika , Lurz Rudi , Lanka Erich 
Published: January 2003

ABSTRACT

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in
trbE
resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in
Escherichia coli
. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.
Traffic at the tmRNA Gene
Authors: Williams Kelly P. 
Published: January 2003

ABSTRACT

A partial screen for genetic elements integrated into completely sequenced bacterial genomes shows more significant bias in specificity for the tmRNA gene (
ssrA
) than for any type of tRNA gene. Horizontal gene transfer, a major avenue of bacterial evolution, was assessed by focusing on elements using this single attachment locus. Diverse elements use
ssrA
; among enterobacteria alone, at least four different integrase subfamilies have independently evolved specificity for
ssrA
, and almost every strain analyzed presents a unique set of integrated elements. Even elements using essentially the same integrase can be very diverse, as is a group with an
ssrA
-specific integrase of the P4 subfamily. This same integrase appears to promote damage routinely at attachment sites, which may be adaptive. Elements in arrays can recombine; one such event mediated by invertible DNA segments within neighboring elements likely explains the monophasic nature of
Salmonella enterica
serovar Typhi. One of a limited set of conserved sequences occurs at the attachment site of each enterobacterial element, apparently serving as a transcriptional terminator for
ssrA
. Elements were usually found integrated into tRNA-like sequence at the 3′ end of
ssrA
, at subsites corresponding to those used in tRNA genes; an exception was found at the non-tRNA-like 3′ end produced by
ssrA
gene permutation in cyanobacteria, suggesting that, during the evolution of new site specificity by integrases, tropism toward a conserved 3′ end of an RNA gene may be as strong as toward a tRNA-like sequence. The proximity of
ssrA
and
smpB
, which act in concert, was also surveyed.
Microarray Analysis of Global Gene Expression in Mucoid
Pseudomonas aeruginosa
Authors: Firoved Aaron M. , Deretic Vojo 
Published: January 2003

ABSTRACT

Pseudomonas aeruginosa
is the dominant pathogen causing chronic respiratory infections in cystic fibrosis (CF). After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of
P. aeruginosa
to the mucoid, exopolysaccharide alginate-overproducing phenotype. The emergence of mucoid
P. aeruginosa
in CF is associated with respiratory decline and poor prognosis. The switch to mucoidy in most CF isolates is caused by mutations in the
mucA
gene encoding an anti-sigma factor. The mutations in
mucA
result in the activation of the alternative sigma factor AlgU, the
P. aeruginosa
ortholog of
Escherichia coli
extreme stress sigma factor σ
E
. Because of the global nature of the regulators of mucoidy, we have hypothesized that other genes, in addition to those specific for alginate production, must be induced upon conversion to mucoidy, and their production may contribute to the pathogenesis in CF. Here we applied microarray analysis to identify on the whole-genome scale those genes that are coinduced with the AlgU sigmulon upon conversion to mucoidy. Gene expression profiles of AlgU-dependent conversion to mucoidy revealed coinduction of a specific subset of known virulence determinants (the major protease elastase gene, alkaline metalloproteinase gene
aprA
, and the protease secretion factor genes
aprE
and
aprF
) or toxic factors (cyanide synthase) that may have implications for disease in CF. Analysis of promoter regions of the most highly induced genes (>40-fold,
P
≤ 10
−4
) revealed a previously unrecognized, putative AlgU promoter upstream of the osmotically inducible gene
osmE
. This newly identified AlgU-dependent promoter of
osmE
was confirmed by mapping the mRNA 5′ end by primer extension. The recognition of genes induced in mucoid
P. aeruginosa
, other than those associated with alginate biosynthesis, reported here revealed the identity of previously unappreciated factors potentially contributing to the morbidity and mortality caused by mucoid
P. aeruginosa
in CF.
Attachment Organelle Formation Represented by Localization of Cytadherence Proteins and Formation of the Electron-Dense Core in Wild-Type and Mutant Strains of
Mycoplasma pneumoniae
Authors: Seto Shintaro , Miyata Makoto 
Published: January 2003

ABSTRACT

Cytadherence proteins of
Mycoplasma pneumoniae
are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division. The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy. In the class I-2 mutant, which has a frameshift mutation in the
hmw2
gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing. However, foci for P90, P40, P30, and P65 were not observed in this mutant. In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused. In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell. These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65. The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core. Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain. HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3. These differences were more pronounced in cytadherence mutants. These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65.
Characterization of the CipA Scaffolding Protein and In Vivo Production of a Minicellulosome in
Clostridium acetobutylicum
Authors: Sabathé Fabrice , Soucaille Philippe 
Published: January 2003

ABSTRACT

The
cipA
gene encoding the
Clostridium acetobutylicum
scaffolding protein CipA was cloned and expressed in
Escherichia coli
. CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains. The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain. Affinity-purified CipA was used in equilibrium-binding experiments to characterize the interaction of CipA with crystalline cellulose. A

K
d

of 0.038 μM and a [C]
max
of 0.43 μmol of CipA bound per g of Avicel were determined. A mini-CipA polypeptide consisting of a CBD3a and two cohesin domains was overexpressed in
C. acetobutylicum
, yielding the in vivo formation of a minicellulosome. This is to our knowledge the first demonstration of the in vivo assembly of a recombinant minicellulosome.
Plasmid DNA Supercoiling and Gyrase Activity in
Escherichia coli
Wild-Type and
rpoS
Stationary-Phase Cells
Authors: Reyes-Domínguez Yazmid , Contreras-Ferrat Gabriel , Ramírez-Santos Jesús , Membrillo-Hernández Jorge , Gómez-Eichelmann M. Carmen 
Published: January 2003

ABSTRACT

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase
rpoS
cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that
rpoS
plays a role in the regulation of plasmid topology during the stationary phase.
The NorM Efflux Pump of
Neisseria gonorrhoeae
and
Neisseria meningitidis
Recognizes Antimicrobial Cationic Compounds
Authors: Rouquette-Loughlin Corinne , Dunham Steven A. , Kuhn Michael , Balthazar Jacqueline T. , Shafer William M. 
Published: January 2003

ABSTRACT

In
Neisseria gonorrhoeae
and
Neisseria meningitidis
, we identified a gene that would encode a protein highly similar to NorM of
Vibrio parahaemolyticus
(Y. Morita et al., Antimicrob. Agents Chemother. 42:1778-1782, 1998). A nonpolar insertional mutation in either the gonococcal or meningococcal
norM
gene resulted in increased bacterial sensitivity to compounds harboring a quaternary ammonium on an aromatic ring (e.g., ethidium bromide, acriflavine hydrochloride, 2-
N
-methylellipticinium, and berberine). The presence of point mutations within the −35 region of a putative
norM
promoter or a likely ribosome binding site resulted in an increased resistance of gonococci and meningococci to the same compounds, as well as to norfloxacin and ciprofloxacin. Structure-activity relationship studies with putative NorM substrates have found that a cationic moiety is essential for NorM recognition.
The High-Pathogenicity Island Is Absent in Human Pathogens of
Salmonella enterica
Subspecies I but Present in Isolates of Subspecies III and VI
Authors: Oelschlaeger T. A. , Zhang D. , Schubert S. , Carniel E. , Rabsch W. , Karch H. , Hacker J. 
Published: January 2003

ABSTRACT

In this study we tested 74
Salmonella
strains of all eight
Salmonella
groups and were able to demonstrate the presence of two high-pathogenicity island types in strains of
Salmonella
groups IIIa, IIIb, and VI. Most high-pathogenicity island-positive isolates produced yersiniabactin under iron-limited conditions and were positive for the high-molecular-weight proteins HMWP1 and HMWP2.
Genetic and Biochemical Studies of Phosphatase Activity of PhoR
Authors: Carmany Daniel O. , Hollingsworth Kristine , McCleary William R. 
Published: January 2003

ABSTRACT

In
Escherichia coli
, PhoR is the histidine kinase of the phosphate regulon. It has been postulated that PhoR may function as a phospho-PhoB phosphatase. Experiments with four precise
phoR
deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain. This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.
Expression of Primary Sigma Factor (PSF) and PSF-Like Sigma Factors in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Tuominen Ilona , Tyystjärvi Esa , Tyystjärvi Taina 
Published: January 2003

ABSTRACT

Large amounts of
sigA
mRNA, encoding the primary σ factor (PSF) in
Synechocystis
sp. strain PCC 6803, accumulated under standard growth conditions, while stress conditions like heat or high salinity led to a rapid decrease in
sigA
mRNA content. The
sigB
,
sigC
,
sigD
, and
sigE
genes, encoding PSF-like σ factors, were under strict physiological control.
Chromosomal Arm Replacement in
Streptomyces griseus
Authors: Uchida Tetsuya , Miyawaki Mariko , Kinashi Haruyasu 
Published: January 2003

ABSTRACT

UV irradiation of
Streptomyces griseus
2247 yielded a new chromosomal deletion mutant, MM9. Restriction and sequencing analysis revealed that homologous recombination between two similar lipoprotein-like open reading frames, which are located 450 and 250 kb from the left and right ends, respectively, caused chromosomal arm replacement. As a result, new 450-kb terminal inverted repeats (TIRs) were formed in place of the original 24-kb TIRs. Frequent homologous recombinations in
Streptomyces
strains suggest that telomere deletions can usually be repaired by recombinational DNA repair functioning between the intact and deleted TIR sequences on the same chromosome.
Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of
Ruminococcus flavefaciens
Authors: Rincon Marco T. , Ding Shi-You , McCrae Sheila I. , Martin Jennifer C. , Aurilia Vincenzo , Lamed Raphael , Shoham Yuval , Bayer Edward A. , Flint Harry J. 
Published: January 2003

ABSTRACT

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe
Ruminococcus flavefaciens
17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in
E. coli
; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in
R. flavefaciens
plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative
R. flavefaciens
bands, including an ∼110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in
R. flavefaciens
.
ISCce
1
and ISCce
2
, Two Novel Insertion Sequences in
Clostridium cellulolyticum
Authors: Maamar Hédia , de Philip Pascale , Bélaich Jean-Pierre , Tardif Chantal 
Published: January 2003

ABSTRACT

Two new insertion sequences, ISCce
1
and ISCce
2
, were found to be inserted into the
cipC
gene of spontaneous mutants of
Clostridium cellulolyticum
. In these insertional mutants, the
cipC
gene was disrupted either by ISCce
1
alone or by both ISCce
1
and ISCce
2
. ISCce
1
is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS
481
and IS
3
families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce
1
. ISCce
2
is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS
256
family. Upon transposition, both ISCce
1
and ISCce
2
generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce
1
is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce
2
was found to be mostly inserted into ISCce
1
. In addition, as neither of the elements was detected in seven other
Clostridium
species, we concluded that they may be specific to the
C. cellulolyticum
strain used.
Characterization of
Helicobacter pylori
Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase
Authors: Mehta Nalini , Olson Jonathan W. , Maier Robert J. 
Published: January 2003

ABSTRACT

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in
Helicobacter pylori
. Here, the two proteins were purified and characterized. HypA bound two Ni
2+
ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants
K
1
and
K
2
for Ni
2+
were 58 and 1.3 μM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of
hypA
(in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 μM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of
hypA
) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.
ATP-Dependent Interactions between
Escherichia coli
Min Proteins and the Phospholipid Membrane In Vitro
Authors: Lackner Laura L. , Raskin David M. , de Boer Piet A. J. 
Published: January 2003

ABSTRACT

Proper placement of the division apparatus in
Escherichia coli
requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg
2+
, whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.