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Antibacterial Activities of Rhodamine B-Conjugated Gelsolin-Derived Peptides Compared to Those of the Antimicrobial Peptides Cathelicidin LL37, Magainin II, and Melittin. Robert Bucki, 2004.The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work . We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can kill the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and the gram-positive organism Streptococcus pneumoniae . RhB linkage to the QRLFQVKGRR sequence in gelsolin was essential for the antibacterial function, since the unconjugated peptide had no effect on the bacteria tested . Because RhB-QRLFQVKGRR (also termed PBP10), its scrambled sequence (RhB-FRVKLKQGQR), and PBP10 synthesized from D-isomer amino acids show similar antibacterial properties, the physical and chemical properties of these derivatives appear to be more important than specific peptide folding for their antibacterial functions . The similar activities of PBP10 and all-D-amino-acid PBP10 also indicate that a specific interaction between RhB derivatives and bacterial proteins is unlikely to be involved in the bacterial killing function of PBP10 . By using a phospholipid monolayer system, we found a positive correlation between the antibacterial function of PBP10, as well as some naturally occurring antibacterial peptides, and the intrinsic surface pressure activity at the hydrophobic-hydrophilic interface . Surprisingly, we observed little or no dependence of the insertion of these peptides into lipid monolayers on the phospholipid composition . These studies show that an effective antimicrobial agent can be produced from a peptide sequence with specificity to a phospholipid not found in bacteria, and comparisons with other antimicrobial agents suggest that the surface activities of these peptides are more important than specific binding to bacterial proteins or lipids for their antimicrobial functions . Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm. Karin Sauer, 2002.Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis . Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations . Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion . Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development . The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins) . When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome) . This difference was higher than when planktonic P . aeruginosa were compared with planktonic cultures of Pseudomonas putida . Las quorum sensing was shown to play no role in early biofilm development but was important in later stages . Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria . These results demonstrate that P . aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth . KtrAB and KtrCD: Two K+ Uptake Systems in Bacillus subtilis and Their Role in Adaptation to Hypertonicity. Gudrun Holtmann, 2003.Recently, a new type of K+ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T . Nakamura, R . Yuda, T . Unemoto, and E . P . Bakker, J . Bacteriol . 180:3491-3494, 1998) . The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane . The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA (ktrA) and ykqB (ktrC) encode homologues to the V . alginolyticus KtrA protein, and yubG (ktrB) and ykrM (ktrD) encode homologues to the V . alginolyticus KtrB protein . We constructed gene disruption mutations in each of the four B . subtilis ktr genes and used this isogenic set of mutants for K+ uptake experiments . Preliminary K+ transport assays revealed that the KtrAB system has a moderate affinity with a Km value of approximately 1 mM for K+, while KtrCD has a low affinity with a Km value of approximately 10 mM for this ion . A strain defective in both KtrAB and KtrCD exhibited only a residual K+ uptake activity, demonstrating that KtrAB and KtrCD systems are the major K+ transporters of B . subtilis . Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD, which occupy different locations on the B . subtilis chromosome, are expressed as single transcriptional units . The amount of K+ in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts . A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective . In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K+ ions from the cells . Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ, opuA, and gsiB genes was not impaired, demonstrating that a high cytoplasmic K+ concentration is not essential for the transcriptional activation of these genes at high osmolarity . Taken together, our data suggest that K+ uptake via KtrAB and KtrCD is an important facet in the cellular defense of B . subtilis against both suddenly imposed and prolonged osmotic stress .
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