Autoinducers extracted from microbial mats reveal a surprising diversity of N-acylhomoserine lactones (AHLs) and abundance changes that may relate to diel pH.

Microbial mats are highly structured and diverse communities, and one of the earliest-known life assemblages. Mat bacteria interact within an environment marked by strong geochemical gradients and fluctuations. We examined natural mat systems for the presence of autoinducers involved in quorum sensing, a form of cell-cell communication. Our results revealed that a diverse array of N-acylhomoserine lactones (AHLs) including C(4)- to C(14)-AHLs, were identified from mat extracts using mass spectrometry (MS), with further confirmation by MS/MS-collision-induced dissociation (CID), and additions of external standards. Microelectrode measurements showed that mats exhibited diel pH fluctuations, ranging from alkaline (pH 9.4) during daytime (net photosynthesis) to acidic (pH 6.8) during darkness (net respiration/fermentation). Under laboratory conditions, AHLs having shorter acyl-chains were degraded within the time frame that daily alkaline pH (> 8.2) conditions exist in mats. Intensive sampling of mats after full day- or night-time incubations revealed that accumulations of extractable shorter-chain AHLs (e.g. C(8)- and C(10)-AHLs) were significantly (P < 0.001) diminished during daytime. Our study offers evidence that stabilities of AHLs under natural conditions may be influenced by the proximal extracellular environment. We further propose that the ancient periodicity of photosynthesis/respiration in mats may potentially drive a mechanism for diel differences in activities of certain autoinducers, and hence bacterial activities mediated through quorum sensing.

Characteristics and turnover of exopolymeric substances in a hypersaline microbial mat.

The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO(3)) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c. 80% of the EPS were turned over in the upper 15-20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5-8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO(3). We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO(3) to precipitate. Therefore, this model explains interactions between EPS and CaCO(3) precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.

Humanized docking system for assembly of targeting drug delivery complexes.

Targeted drug delivery requires 'loading' drugs onto targeting proteins. Traditional technologies for loading drugs rely on chemical conjugation of drugs or drug carriers to targeting proteins. An alternative approach might rely on assembly of targeting complexes using a docking system that includes two components: a 'docking' tag fused to a targeting protein, and a 'payload' module containing an adapter protein for non-covalent binding to the docking tag. We describe here a fully humanized adapter/docking tag system based on non-covalent interaction between two fragments of human pancreatic RNase I. A 15 amino acid long N-terminal fragment of RNase I designed to serve as a docking tag, was fused to the N-terminus of human vascular endothelial growth factor that served as a targeting protein. An 18-125 and an 18-127 amino acid long fragments of RNase I were engineered, expressed and refolded into active conformations to serve as adapter proteins. Interactions between the targeting and adapter proteins were characterized using enzymatic analysis and surface plasmon resonance. Targeting DNA delivery complexes were assembled, characterized by dynamic light scattering, and found to be very effective in receptor-mediated DNA delivery.

Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery.

High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery. The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein. To avoid random chemical crosslinking of drug carriers to S-protein, we constructed a mutant 16-124aa fragment of RNase A in which 122ala is replaced with a cysteine residue. The mutant and the corresponding wild type fragments expressed in Escherichia coli are refolded into functional conformations only in the presence of S-peptide. After the removal of S-peptide, both fragments retain the ability to bind S-peptide and S-peptide-tagged proteins. The 122cys residue in the mutant fragment is available for site-specific conjugation.

Molecular vehicles for targeted drug delivery.

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.