Borate Diester Research Articles

Anti-quorum sensing potential of the mangrove Rhizophora annamalayana

The present study was carried out to assess the anti-quorum sensing (anti-QS) activity of bark extract obtained from the mangrove plant Rhizophora annamalayana Kathir. against Gram-negative bacteria. In microtitre plate assay, the bark extract at a concentration of 1mg/ml inhibited the QS-dependent violacein production in Chromobacterium violaceum ATCC 12472. Further, the QS-dependent bioluminescence production in the aquatic bacterial pathogen Vibrio harveyi MTCC 3438 was also reduced to the level of 99% when treated with the same concentration of the extract. Gas chromatography-mass spectrum analysis identified the presence of seven different chemical constituents, 1H-purin-6-amine, cycloheptasiloxane, cyclooctasiloxane, cyclononasiloxane, cyclononasiloxane octadecamethyl, cyclodecasiloxane eicosamethyl and 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy)tetrasiloxane. The molecular docking analysis of the identified compounds revealed that the compounds cyclononasiloxane octadecamethyl and cyclodecasiloxane eicosamethyl exhibited the best docking energy with the QS receptors of C. violaceum and V. harveyi with that of the natural ligand N -hexanoyl- L -homoserine lactone (C6-HSL) and furanosyl borate diester (AI-2). Similarly, another compound 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy)tetrasiloxane showed best docking energy only against C6-HSL. Thus, the results of the present study divulge the activity of R. annamalayana bark extract to interfere with bacterial QS.

Identification of putative rhamnogalacturonan-II specific glycosyltransferases in Arabidopsis using a combination of bioinformatics approaches.

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.

Quorum sensing in the probiotic bacterium Escherichia coli Nissle 1917 (Mutaflor) - evidence that furanosyl borate diester (AI-2) is influencing the cytokine expression in the DSS colitis mouse model.

Background“Quorum sensing” (QS) is the phenomenon which allows single bacterial cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) and AI-2 for the interspecies communication. Aim of this study was to investigate QS of Escherichia coli Nissle 1917 (Mutaflor).ResultsWhile E. coli Nissle is producing AI-2 in a density dependent manner, no AI-1 was produced. To study the effect of AI-2 in the DSS (dextran sulphate sodium) induced mouse model of acute colitis, we silenced the corresponding gene luxS by intron insertion. The mutant bacterium E. coli Nissle::luxS was equally effective in colonizing the colon and the mutation turned out to be 100% stable during the course of the experiment. Isolating RNA from the colon mucosa and performing semiquantitative RT PCR, we were able to show that the expression of the pro-inflammatory cytokine IFN-y was suppressed in mice being infected with the E. coli Nissle wild type. Mice infected with the E. coli Nissle::luxS mutant showed a suppressed expression of IL-10 compared to uninfected mice, while the expression of the pro-inflammatory cytokines IL-6 and TNF-α was higher in these mice. The expression of mBD-1 was suppressed in mice being infected with the mutant in comparison to the mice not infected or infected with the wild type. No differences were seen in the histological examination of the colon sections in the different groups of mice.ConclusionsE. coli Nissle is producing AI-2 molecules, which are influencing the expression of cytokines in the mucosa of the colon in the DSS mice. However, if QS has a direct influence on the probiotic properties of E. coli Nissle remains to be elucidated.

The Response of Type 2 Quorum Sensing in Klebsiella pneumoniae to a Fluctuating Culture Environment

Bacterial cells communicate with one another using chemical signaling molecules. This phenomenon is termed quorum sensing (QS). QS in Klebsiella pneumoniae is mediated by the synthesis of interspecies autoinducer 2 (AI-2), a furanosyl borate diester molecule. The response of Type 2 QS to environmental cues such as carbon sources, the initial pH of the medium, and boracic acid was investigated in the present study using a Vibrio harveyi BB170 reporter assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 3.0% glucose can stimulate K. pneumoniae to produce more AI-2, with a four times increase in activity compared with that of the control culture. According to our previous research, Type 2 QS in K. pneumoniae is luxS dependent. Therefore, the close relationship between glucose concentration and luxS transcription level was confirmed with qRT-PCR technology. The results show that the response of QS to a fluctuating glucose concentration was observed as a change in the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 30.0 g/L. At the same time, AI-2 production was also slightly impacted by the low initial pH. It is noteworthy that the addition of boracic acid at microdosage (0.1 g/L) can also induce AI-2 synthesis. Presumably, in K. pneumoniae, the 4,5-dihydroxy-2,3-pentanedione cyclizes by the addition of borate and loss of water, is hydrated, and is converted to the final AI-2 signaling molecule.

Can Bacteria Evolve Resistance to Quorum Sensing Disruption?

Traditional treatment of bacterial infections relies heavily on the use of antibacterial compounds that either kill bacteria (bactericidal) or inhibit their growth (bacteriostatic). Typically, the targets for the main conventional antibiotics are essential cellular processes such as bacterial cell wall biosynthesis, bacterial protein synthesis, and bacterial DNA replication and repair. However, resistance to these drugs arises and spreads very rapidly, even to such an extent that bacteria have been identified that are simultaneously resistant to all available antibiotics [1]. The increasing occurrence of resistant bacteria gradually renders antibiotics ineffective in treating infections and has enormous human and economic consequences worldwide. As a result, the identification of novel drug targets and the development of novel therapeutics constitute an important area of current scientific research. An alternative to killing or inhibiting growth of pathogenic bacteria is the specific attenuation of bacterial virulence, which can be attained by targeting key regulatory systems that mediate the expression of virulence factors. One of the target regulatory systems is quorum sensing (QS), or bacterial cell-to-cell communication. QS is a mechanism of gene regulation in which bacteria coordinate the expression of certain genes in response to the presence or absence of small signal molecules (Figure 1). Figure 1 General scheme of a quorum sensing system. Quorum Sensing: Bacterial Cell-to-Cell Communication QS was first discovered in the marine bacterium Vibrio fischeri and was thought to be restricted to only a limited series of species. Later on, similar systems were found to be present in many other Gram-negative bacteria. These Gram-negative bacteria use acylated homoserine lactones (AHLs) as signal molecules (for a review see [2]). AHLs are typically produced by a homolog of V. fischeri LuxI and detected by a homolog of V. fischeri LuxR. In addition to the AHL-mediated systems in Gram-negative bacteria, some Gram-positive bacteria also regulate a variety of processes by QS. The QS systems of Streptococcus pneumoniae, Bacillus subtilis, and Staphylococcus aureus, for instance, have been extensively studied (for a review see [3]). A different kind of QS system is found in vibrios. These bacteria use multichannel QS systems in which different types of signal molecules are produced. The signal molecules are detected at the cell surface by membrane-bound, two-component receptor proteins that feed a common phosphorylation/dephosphorylation signal transduction cascade (for a review on QS in vibrios, see [4]). One of the signals produced by vibrios is the so-called autoinducer 2 (AI-2), a furanosyl borate diester [5]. AI-2 activity has been detected in many different species (Gram-negative as well as Gram-positive), although its function as a signal is not generally accepted for all species (for a detailed discussion see [6]). The language of bacteria seems to be even more diversified as new QS systems, using different types of signal molecules, are still being discovered [7].

Quorum sensing and bacterial biofilms

This review describes the chemistry of the bacterial biofilms including the chemistry of their constituents and signalling compounds that mediate or inhibit the formation of biofilms. Systems are described with special emphasis, in which quorum sensing molecules (autoinducers) trigger the formation of biofilms. In the first instance, N-acyl-L-homoserine lactones (AHLs) are the focus of this review, whereas the inter-species signal known as furanosyl borate diester and peptide autoinducers used by Gram-positive bacteria are not discussed in detail. Since the first discovery of an AHL autoinducer from Vibrio fischeri a large and further increasing number of different AHL structures from Gram-negative bacteria have been identified. This review gives a summary of all known AHL autoinducers and producing bacterial species. A few systems are discussed, where biofilm formation is suppressed by enzymatic degradation of AHL molecules or interference of secondary metabolites from other species with the quorum sensing systems of communicating bacteria. Finally, the multi-channel quorum sensing system, the intracellular downstream processing of the signal, and the resulting response of whole populations including biofilm formation are discussed for the Vibrio genus that has been extensively investigated.

Pseudomonas fragi strains isolated from meat do not produce N-acyl homoserine lactones as signal molecules.

Quorum sensing (QS) is a signalling mechanism through which bacteria cellular functions are modified to promote access to nutrients and more favorable environmental niches. The frequent occurrence of Pseudomonas spp. in fresh and spoiled meat may involve enhanced gene expression regulated by QS. Several Pseudomonas spp. produce different N-acyl homoserine lactone (AHL) signal molecules. Meat spoilage during aerobic, refrigerated storage is often associated with the presence of Pseudomonas fragi. As with other Pseudomonas species in natural habitats, the dominance and activities of P. fragi in meat may be regulated by QS. In this study, five biosensor strains were used to detect AHL production on three different media by 72 different P. fragi strains isolated from fresh and spoiled meat. Positive and negative AHL-producing strains were used to verify the assays. None of the strains produced detectable quantities of AHLs, even when concentrated cell-free culture supernatants were assayed, nor did exogenous lactones increase biofilm formation in P. fragi strains. However, all isolates produced furanosyl borate diesters (type II autoinducers; AI-2) when tested using the bioluminescent biosensor strain of Vibrio harveyi (BB170). The production of AI-2 was presumed to be of metabolic origin even though Pseudomonas spp. have not been shown to harbor the luxS gene. Thus, the efficient development of P. fragi in fresh meat is not regulated by an AHL-mediated QS system. The mechanism of AI-2 production and its possible role in spoilage dynamics needs further study.

Can Infectious Biofilm be Controlled by Blocking Bacterial Communication?

Many bacteria grow on surfaces forming biofilms but often high dosages of antibiotics can not clear infectious biofilms. Biofilms are controlled by a process called quorum-sensing triggered by species-specific, small, diffusible autoinducers (N-acyl homoserine lactones (AHL), small peptides or a furanosyl borate diester). Several herbal, animal and microbial extracts possess quorum-quenching activity but only few active compounds and synthetic analogues are known. However, quorum-sensing seems not to be the only way to control biofilms. Bacteria have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only few compounds responsible for the signalling of these processes are known but may open a completely novel line of biofilm control. Furthermore, controlling the attachment of bacterial by inhibiting the formation of pili is another option and some inhibitors have already been characterized. In this article different approaches to control bacterial biofilms are discussed together with the balance between biofilm formation and virulence.

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation: Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.

Vibrio vulnificus produces quorum sensing signals of the AHL-class

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N-butanoyl-homoserine-lactone (C(4)-HL) by both reporter strains and by HPLC-high-resolution MS. C(4)-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain-heart infusion) and its production was enhanced when blood factors were added to MSWYE. C(4)-HL was detected in vivo, in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD.

Cloning and characterizations of the Serratia marcescens metK and pfs genes involved in AI-2-dependent quorum-sensing system

Serratia marcescens utilizes two types of quorum-sensing signal molecules: N-acyl homoserine lactones and furanosyl borate diester (AI-2). S-adenosylmethionine synthetase (METK), S-adenosylhomocysteine nucleosidase (PFS), and S-ribosylhomocysteinase (LUXS) are three key enzymes in the biosynthetic pathway leading to AI-2 production. The sequence of luxS gene was published at NCBI (Accession number: EF164926). So in this study, Serratia marcescens metK and pfs genes were successfully cloned with inverse PCR. The results show that the ORF lengths of metK and pfs are 1155 and 702 bp, and encode proteins of 384 and 233 residues. Their molecular weights and isoelectric points are 41.85 kD and 5.50; 27.67 kD and 5.56, which are acidic proteins judging from the calculated pI values. Expression products of two genes with pET28a((+)) system exhibited molecular weights in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis comparable with a theoretical estimation. The sequences of these two genes were conferred China patent application numbers CN 200710048016.X and CN 200710048015.5, respectively.

Oxazaborolidine derivatives inducing autoinducer-2 signal transduction in Vibrio harveyi

The bioluminescence of the marine bacterium Vibrio harveyi is controlled by quorum sensing. This effect is mediated by production, accumulation, and auto-detection of the species-specific autoinducer 1 (AI-1), autoinducer 2 (AI-2), and the V. cholerae autoinducer 1 (CAI-1). The V. harveyi AI-2 was recently identified as furanosyl borate diester. We synthesized several oxazaborolidine derivatives that chemically resemble the structure of AI-2. Five oxazaborolidine derivatives (BNO-1 to BNO-5) were tested, however only BNO-1 (3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaborolidine), and BNO-5 (2-butyl-3,4-dimethyl-5-phenyl-1,3,2-oxazaborolidine) strongly induced V. harveyi bioluminescence in V. harveyi mutant (BB170) lacking sensor 1. A dose-dependent relationship between those oxazaborolidine derivatives and bioluminescence induction was observed with this V. harveyi strain (BB170). BNO-1 and BNO-5 did not affect V. harveyi BB886 lacking sensor 2. Using a mutant strain which produces neither AI-1 nor AI-2 ( V. harveyi MM77) we showed that the presence of spent medium containing AI-2 is essential for BNO-1 and BNO-5 activity. This effect was similar when introducing the spent medium and the BNOs together or at a 3-h interval. A comparable induction of bioluminescence was observed when using synthetic DPD (pre-AI-2) in the presence of BNO-1 or BNO-5. The mode of action of BNO-1 and BNO-5 on bioluminescence of V. harveyi is of a co-agonist category. BNO-1 and BNO-5 enhanced AI-2 signal transduction only in the presence of AI-2 and only via sensor 2 cascade. BNO-1 and BNO-5 are the first oxazaborolidines reported to affect AI-2 activity. Those derivatives represent a new class of borates which may become prototypes of novel agonists of quorum sensing mediated by AI-2 in V. harveyi.

The major Vibrio cholerae autoinducer and its role in virulence factor production

Vibrio cholerae, the causative agent of the human disease cholera, uses cell-to-cell communication to control pathogenicity and biofilm formation. This process, known as quorum sensing, relies on the secretion and detection of signalling molecules called autoinducers. At low cell density V. cholerae activates the expression of virulence factors and forms biofilms. At high cell density the accumulation of two quorum-sensing autoinducers represses these traits. These two autoinducers, cholerae autoinducer-1 (CAI-1) and autoinducer-2 (AI-2), function synergistically to control gene regulation, although CAI-1 is the stronger of the two signals. V. cholerae AI-2 is the furanosyl borate diester (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. Here we describe the purification of CAI-1 and identify the molecule as (S)-3-hydroxytridecan-4-one, a new type of bacterial autoinducer. We provide a synthetic route to both the R and S isomers of CAI-1 as well as simple homologues, and we evaluate their relative activities. Synthetic (S)-3-hydroxytridecan-4-one functions as effectively as natural CAI-1 in repressing production of the canonical virulence factor TCP (toxin co-regulated pilus). These findings suggest that CAI-1 could be used as a therapy to prevent cholera infection and, furthermore, that strategies to manipulate bacterial quorum sensing hold promise in the clinical arena.

A LuxP-FRET-Based Reporter for the Detection and Quantification of AI-2 Bacterial Quorum-Sensing Signal Compounds

Various bacterial species produce and monitor low-molecular weight signaling molecules that regulate specific sets of genes in a population density-dependent manner. This process is known as quorum sensing (QS). To date, the detection of QS signaling molecules from Gram-negative bacteria has relied primarily on bacterial reporter strains. These bioassays are subject to substantial interference by compounds that affect the growth and metabolism of the reporter strains. In addition, the sensitivity of reporter strains to QS signaling molecules is population density-dependent. Here, we describe the development of an in vitro assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer 2 (AI-2), QS molecules. The sensor is based on ligand binding-induced changes in fluorescence resonance energy transfer (FRET) between a cyan and yellow variant of GFP fused to the termini of the BAI-2 receptor, LuxP. Unexpectedly, the addition of synthetic BAI-2 to the purified biosensor induces a decrease in the level of FRET between the terminal fluorophores. Several lines of evidence, including mutation of the ligand binding sites, indicate that the observed FRET changes are BAI-2-dependent. The FRET-based BAI-2 biosensor responded to the addition of culture filtrates from wild-type Vibrio harveyi but exhibited no response to culture filtrates from V. harveyi mutants defective in BAI-2 synthesis. The sensitivity of the biosensor to BAI-2 (apparent Kd = 270 nM) was similar to that of BAI-2 bioassay systems. The limitations of microbial bioassay systems and the advantages and potential applications for the FRET-based BAI-2 biosensor are discussed.

Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay

Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe(3+) within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 microM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of approximately 30% in a reliable and reproducible way.

Boric acid binding studies with diol containing polyethylenimines as determined by 11B NMR spectroscopy

AbstractThree water‐soluble polymers incorporating increasing levels of 2,3‐dihydroxy propyl attached to polyethylenimine (PEI) backbone were synthesized, characterized by NMR, and investigated for their ability to bind boric acid (BA). 11B NMR spectroscopy showed that BA interacted with the polymeric 2,3‐dihydroxy propyls by forming borate monoester and borate diesters in the boron concentration range of 100–1000 ppm and at 0.0775M polymer. Borate monoester species predominated for low functionalization levels (33% of the PEI amines functionalized), whereas borate diester species dominated for the higher functionalized polymers (66–100% of the PEI amines functionalized). All three polymers showed that 100% of the BA was bound as a mixture of borate mono‐ and diesters at 100‐ppm boron. The overall best performer based on total borate ester formation was the 2/3‐PEI, with a binding Kd of 631 at 200 ppm boron. Borate ion concentration was measured from the 11B NMR chemical shift of the BA/borate peak and it decreased as 1/3‐PEI > 2/3‐PEI > 3/3‐PEI. Variable temperature 11B NMR showed drastic reduction of borate ester species at 65°C. Thus, PEI polymers, as the ones investigated in this work, are reasonable candidates for the selective recovery and recycle of BA. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4411–4418, 2006

AI-1 Influences the Kinase Activity but Not the Phosphatase Activity of LuxN of Vibrio harveyi

The Gram-negative bacterium Vibrio harveyi produces and responds to three autoinducers, AI-1, AI-2, and CAI-1 to regulate cell density dependent gene expression by a process referred to as quorum sensing. The concentration of the autoinducers is sensed by three cognate hybrid sensor kinases, and information is channeled via the HPt protein LuxU to the response regulator LuxO. Here, a detailed biochemical study on the enzymatic activities of the membrane-integrated hybrid sensor kinase LuxN, the sensor for N-(d-3-hydroxybutanoyl)homoserine lactone (AI-1), is provided. LuxN was heterologously overproduced as the full-length protein in Escherichia coli. LuxN activities were characterized in vitro and are an autophosphorylation activity with an unusually high ATP turnover rate, stable LuxU phosphorylation, and a slow phosphatase activity with LuxU approximately P as substrate. The presence of AI-1 affected the kinase but not the phosphatase activity of LuxN. The influence of AI-1 on the LuxN--> LuxU signaling step was monitored, and in the presence of AI-1, the kinase activity of LuxN, and hence the amount of LuxU approximately P produced, were significantly reduced. Half-maximal inhibition of kinase activity by AI-1 occurred at 20 mum. Together, these results indicate that AI-1 directly interacts with LuxN to down-regulate its autokinase activity and suggest that the key regulatory step of the AI-1 quorum sensing system of Vibrio harveyi is AI-1-mediated repression of the LuxN kinase activity.

Controlling Biofilms of Gram-Positive Pathogenic Bacteria

Many bacteria can form aggregates on interfaces, called biofilms, where they are much more protected against toxic agents such as antibiotics or antibodies. Bacteria organized in biofilms are therefore very difficult to control and often even high dosages of antibiotics cannot clear infectious biofilms. To form biofilms bacteria have to start a complex genetic program to switch from planktonic to sessile lifestyle. This starts with the determination of their cell density, a process called quorum sensing, triggered by small, water soluble molecules, so called autoinducers. From Gram-positive bacteria several small peptides are known, many of them thiolactones. More recently another group of compounds was discovered probably formed from ribose-homocysteine and the first autoinducer-II identified is a furanosyl borate diester. While small peptides are found to trigger the intraspecies communication autoinducer-II is assumed to be used for communication at the interspecies level. Taking the lead structure from these peptides several derivatives have been developed which prevent biofilm formation in many Gram-positive bacteria, including Staphylococcus aureus. Some of these compounds are already in clinical studies. In this review the different approaches to control bacterial biofilms are discussed together with the difficulties arising from the species-specificity of the autoinducers.