Reconstructing promoter activity from Lux bioluminescent reporters.

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

Pilot Evaluation of S-(3-[18F]Fluoropropyl)-D-Homocysteine and O-(2-[18F]Fluoroethyl)-D-Tyrosine as Bacteria-Specific Radiotracers for PET Imaging of Infection.

PURPOSE: There is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled D-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of D-tyrosine and D-methionine, O-(2-[18F]fluoroethyl)-D-tyrosine (D-[18F]FET) and S-(3-[18F]fluoropropyl)-D-homocysteine (D-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection. PROCEDURES: D-[18F]FET and D-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of D-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG. RESULTS: In vitro uptake of both D-[18F]FET and D-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for D-[18F]FPHCys than D-[18F]FET. Blocking experiments with non-radioactive D-[19F]FPHCys confirmed specificity of uptake. In vivo, D-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation. CONCLUSIONS: D-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled D-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

Enterobacterial tumor colonization in mice depends on bacterial metabolism and macrophages but is independent of chemotaxis and motility.

Despite promising results and increasing attention in bacterial cancer therapy, surprisingly little is known about initial tumor colonization and the interaction between bacteria and surrounding tumor tissue. Here, we analyzed the role of chemotaxis, motility, and metabolism both in Escherichia coli and Salmonella enterica serovar Typhimurium strains upon intravenous injection into tumor-bearing mice. In contrast to previous models, we found that chemotaxis and motility do not play a significant role in tumor colonization and bacterial distribution within the tumor. Rather, the whole colonization and intratumoral migration process seems to be a passive mechanism that is influenced by the reticuloendothelial system of the host, by the tumor microenvironment and by the bacterial metabolism. These conclusions were supported by experimental data demonstrating that disruption of the basic branch of the aromatic amino acid biosynthetic pathway and depletion of macrophages, in contrast to flagellar mutations, led to significant changes in bacterial accumulation in tumors of live mice.

Ultrasound-mediation of self-illuminating reporters improves imaging resolution in optically scattering media.

In vivo imaging of self-illuminating bio-and chemiluminescent reporters is used to observe the physiology of small animals. However, strong light scattering by biological tissues results in poor spatial resolution of the optical imaging, which also degrades the quantitative accuracy. To overcome this challenging problem, focused ultrasound is used to modulate the light from the reporter at the ultrasound frequency. This produces an ultrasound switchable light 'beacon' that reduces the influence of light scattering in order to improve spatial resolution. The experimental results demonstrate that apart from light modulation at the ultrasound frequency (AC signal at 3.5 MHz), ultrasound also increases the DC intensity of the reporters. This is shown to be due to a temperature rise caused by insonification that was minimized to be within acceptable mammalian tissue safety thresholds by adjusting the duty cycle of the ultrasound. Line scans of bio-and chemiluminescent objects embedded within a scattering medium were obtained using ultrasound modulated (AC) and ultrasound enhanced (DC) signals. Lateral resolution is improved by a factor of 12 and 7 respectively, as compared to conventional CCD imaging. Two chemiluminescent sources separated by ~10 mm at ~20 mm deep inside a 50 mm thick chicken breast have been successfully resolved with an average signal-to-noise ratio of approximately 8-10 dB.

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria.

BACKGROUND: The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria. RESULTS: Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning. CONCLUSION: The residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.

Synthesis, in Vitro Evaluation, and Radiolabeling of Fluorinated Puromycin Analogues: Potential Candidates for PET Imaging of Protein Synthesis.

There is currently no ideal radiotracer for imaging of protein synthesis rate (PSR) by positron emission tomography (PET). Existing fluorine-18-labeled amino acid-based radiotracers predominantly visualize amino acid transporter processes, and in many cases they are not incorporated into nascent proteins at all. Others are radiolabeled with the short-half-life positron emitter carbon-11, which is rather impractical for many PET centers. Based on the puromycin (6) structural manifold, a series of 10 novel derivatives of 6 was prepared via Williamson ether synthesis from a common intermediate. A bioluminescence assay was employed to study their inhibitory action on protein synthesis, which identified the fluoroethyl analogue 7b as a lead compound. The fluorine-18 analogue was prepared via nucleophilic substitution of the corresponding tosylate precursor in a modest radiochemical yield of 2 ± 0.6% with excellent radiochemical purity (>99%) and showed complete stability over 3 h at ambient temperature.

[(18)F]FDG-6-P as a novel in vivo tool for imaging staphylococcal infections.

BACKGROUND: Management of infection is a major clinical problem. Staphylococcus aureus is a Gram-positive bacterium which colonises approximately one third of the adult human population. Staphylococcal infections can be life-threatening and are frequently complicated by multi-antibiotic resistant strains including methicillin-resistant S. aureus (MRSA). Fluorodeoxyglucose ([(18)F]FDG) imaging has been used to identify infection sites; however, it is unable to distinguish between sterile inflammation and bacterial load. We have modified [(18)F]FDG by phosphorylation, producing [(18)F]FDG-6-P to facilitate specific uptake and accumulation by S. aureus through hexose phosphate transporters, which are not present in mammalian cell membranes. This approach leads to the specific uptake of the radiopharmaceutical into the bacteria and not the sites of sterile inflammation. METHODS: [(18)F]FDG-6-P was synthesised from [(18)F]FDG. Yield, purity and stability were confirmed by RP-HPLC and iTLC. The specificity of [(18)F]FDG-6-P for the bacterial universal hexose phosphate transporter (UHPT) was confirmed with S. aureus and mammalian cell assays in vitro. Whole body biodistribution and accumulation of [(18)F]FDG-6-P at the sites of bioluminescent staphylococcal infection were established in a murine foreign body infection model. RESULTS: In vitro validation assays demonstrated that [(18)F]FDG-6-P was stable and specifically transported into S. aureus but not mammalian cells. [(18)F]FDG-6-P was elevated at the sites of S. aureus infection in vivo compared to uninfected controls; however, the increase in signal was not significant and unexpectedly, the whole-body biodistribution of [(18)F]FDG-6-P was similar to that of [(18)F]FDG. CONCLUSIONS: Despite conclusive in vitro validation, [(18)F]FDG-6-P did not behave as predicted in vivo. However at the site of known infection, [(18)F]FDG-6-P levels were elevated compared with uninfected controls, providing a higher signal-to-noise ratio. The bacterial UHPT can transport hexose phosphates other than glucose, and therefore alternative sugars may show differential biodistribution and provide a means for specific bacterial detection.

The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress.

Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli. We measured the response to zinc of the main specific zinc import and export systems in the wild-type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: first, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments, we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesize that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments.

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe.

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.

Microbial dynamics and flagellate grazing during transition to winter in Lakes Hoare and Bonney, Antarctica.

The planktonic microbial communities of Lakes Hoare and Bonney were investigated during transition into winter. We hypothesized that the onset of darkness induces changes in the functional role of autotrophic and heterotrophic microplankton. Bacteria decreased in Lake Hoare during March-April, while in Lake Bonney bacterial abundances varied. Heterotrophic nanoflagellates (HNAN), phototrophic nanoflagellates (PNAN) and ciliates showed no marked decline with the onset of winter. PNAN outnumbered HNAN in both lakes. Grazing rates of HNAN in Lake Hoare ranged up to 30.8 bacteria per cell day(-1). The HNAN community grazed between 3.74 and 36.6 ng of bacterial carbon day(-1). Mixotrophic PNAN had grazing rates up to 15.2 bacteria per cell day(-1), and their daily community grazing exceeded bacterial production. In Lake Bonney East, PNAN grazing rates ranged up to 12.48 bacteria per cell day(-1) and in Lake Bonney West up to 8.16 bacteria per cell day(-1). As in Lake Hoare, the mixotrophic PNAN grazing rates (up to 950 ng C day(-1)) usually exceeded bacterial production. HNAN grazing rates were generally similar to those in Lake Hoare. As winter encroaches, these lakes move progressively towards heterotrophy and probably function during the winter, enabling populations to enter the short austral summer with actively growing populations.

Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli.

BACKGROUND: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. METHODS AND FINDINGS: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. CONCLUSIONS: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.

Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice.

Specific colonization of solid tumors by bacteria opens the way to novel approaches in both tumor diagnosis and therapy. However, even non-pathogenic bacteria induce responses by the immune system, which could be devastating for a tumor bearing patient. As such effects are caused e.g., by the lipid A moiety of the lipopolysaccharide, a msbB-mutant of the probiotic E. coli Nissle 1917 strain was investigated. Bacteria of the mutant strain did not show any growth defects in culture media when compared to wild-type E. coli Nissle 1917 but were unable to myristoylate lipid A, had less toxic effects on immunocompetent BALB/c mice, and were still able to specifically colonize tumors. Therefore, the modification of lipid A could result in bacterial strains that might be better suited for diagnosis and therapy of tumors than the corresponding wild-type strains, even if those are not considered pathogenic or are of probiotic background.

The filter-feeding ciliates Colpidium striatum and Tetrahymena pyriformis display selective feeding behaviours in the presence of mixed, equally-sized, bacterial prey.

This study examined whether two ciliates could discriminate between equally-sized bacterial prey in mixture and if so, how selectivity might benefit the ciliate population. Live Klebsiella aerogenes, K. ozaenae and Escherichia coli, expressing different coloured fluorescent proteins, were cultured in such a way as to provide populations containing equally-sized cells (to prevent size-selective grazing taking place) and these prey were fed to each ciliate in 50:50 mixtures. Colpidium striatum selected K. aerogenes over K. ozaenae which itself was selected over E. coli. Tetrahymena pyriformis showed no selectivity between K. aerogenes and E. coli but K. aerogenes was selected over K. ozaenae while E. coli was not. This apparent selection of K. aerogenes over K. ozaenae was sustained in ciliate populations with different feeding histories and when K. aerogenes comprised only 20% of the prey mixture, suggesting possible optimal foraging behaviour. The metabolic benefits for selecting K. aerogenes were identified as possibly being an increase in cell biovolume and yield for C. striatum and T. pyriformis, respectively. The mechanism by which these ciliates selected specific bacterial cells in mixture is currently unknown but the use of live fluorescent bacteria, in prey mixtures, offers an exciting avenue for further investigation of selective feeding by protozoa.

Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice.

Systemic administration of microorganisms into tumor-bearing mice revealed preferential accumulation in tumors in comparison to clearance in organs such as spleen and liver. Here we compared the efficiency of tumor-specific colonization of pathogenic Salmonella typhimurium strains 14028 and SL1344 to the enteroinvasive Escherichia coli 4608-58 strain and to the attenuated Salmonella flexneri 2a SC602 strain, as well as to the uropathogenic E. coli CFT073, the non-pathogenic E. coli Top10, and the probiotic E. coli Nissle 1917 strain. All strains colonized and replicated in tumors efficiently each resulting in more than 1 x 10(8) colony-forming units per gram tumor tissue. Colonization of spleen and liver were significantly lower when E. coli strains were used in comparison to S. typhimurium and the non-pathogenic strains did not colonize those organs at all. Further investigation of E. coli Nissle 1917 showed that no drastic differences in colonization and amplification were seen when immunocompetent and immunocompromised animals were used, and we were able to show that E. coli Nissle 1917 replicates at the border of live and necrotic tumor tissue. We also demonstrated exogenously applied L-arabinose-dependent gene activation in colonized tumors in live mice. These findings will prepare the way for bacterium-mediated controlled protein delivery to solid tumors.

Bacterial community structure and location in Stilton cheese.

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.

Demonstration of antifreeze protein activity in Antarctic lake bacteria.

Antifreeze proteins (AFPs) are a structurally diverse group of proteins that have the ability to modify ice crystal structure and inhibit recrystallization of ice. AFPs are well characterized in fish and insects, but very few bacterial species have been shown to have AFP activity to date. Thirty eight freshwater to hypersaline lakes in the Vestfold Hills and Larsemann Hills of Eastern Antarctica were sampled for AFPs during 2000. Eight hundred and sixty six bacterial isolates were cultivated. A novel AFP assay, designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 of the cultures. Subsequent analysis of the putative positive isolates showed 19 isolates with significant recrystallization inhibition (RI) activity. The 19 RI active isolates were characterized using ARDRA (amplified rDNA restriction analysis) and 16S rDNA sequencing. They belong to genera from the alpha- and gamma-Proteobacteria, with genera from the gamma-subdivision being predominant. The 19 AFP-active isolates were isolated from four physico-chemically diverse lakes. Ace Lake and Oval Lake were both meromictic with correspondingly characteristic chemically stratified water columns. Pendant Lake was a saline holomictic lake with different chemical properties to the two meromictic lakes. Triple Lake was a hypersaline lake rich in dissolved organic carbon and inorganic nutrients. The environments from which the AFP-active isolates were isolated are remarkably diverse. It will be of interest, therefore, to elucidate the evolutionary forces that have led to the acquisition of functional AFP activity in microbes of the Vestfold Hills lakes and to discover the role the antifreezes play in these organisms.

Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence.

Clp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature-tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus. Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325-4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall-associated proteins. In the mutant cells, the amount of alpha-haemolysin (hla) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum-sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.

Conservation, surface exposure, and in vivo expression of the Frp family of iron-regulated cell wall proteins in Staphylococcus aureus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic Staphylococcus aureus cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated frpA and frpB, respectively. Studies with an S. aureus fur mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative S. aureus membrane protein, FrpC. frpC is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of S. aureus under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of S. aureus to plastic in vitro. Binding of S. aureus to microtiter wells was found to be iron regulated, and iron-restricted S. aureus containing antisense frpA or frpAB but not frpB constructs showed reduced binding compared to vector construct controls.

LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone.

Many bacteria produce extracellular molecules which function in cell-to-cell communication. One of these molecules, autoinducer 2 (AI-2), was first described as an extracellular signal produced by Vibrio harveyi to control luciferase expression. Subsequently, a number of bacteria have been shown to possess AI-2 activity in their culture supernatants, and bear the luxS gene product, which is required for AI-2 synthesis. In Porphyromonas gingivalis, luxS and pfs, encoding a 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH'ase), form an operon, suggesting that S-adenosylhomocysteine (SAH) or 5'-methylthioadenosine (MTA) serves as a substrate for AI-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5alpha (which carries a luxS frame-shift mutation) were capable of generating AI-2 activity upon addition of SAH, but not MTA. S-Ribosyl-homocysteine (RH) derived from SAH also served as a substrate in E. coli MG1655 extracts. RH-supplemented cell-free extracts of Pseudomonas aeruginosa, a bacterium that lacks luxS, only generated AI-2 activity following the introduction of a plasmid containing the Por. gingivalis pfs-luxS operon. In addition, defined in vitro systems consisting of the purified LuxS proteins from Por. gingivalis, E. coli, Neisseria meningitidis or Staphylococcus aureus converted RH to homocysteine and a compound that exhibits AI-2 activity.4-Hydroxy-5-methyl-3(2H)-furanone was identified by mass spectrometry analysis as a major product formed in this in vitro reaction. In E. coli MG1655, expression of T3SH [the bacteriophage T3 S-adenosylmethionine (SAM) hydrolase] significantly reduced AI-2 activity in culture supernatants, suggesting that AI-2 production is limited by the amount of SAH produced in SAM-dependent transmethylase reactions. The authors suggest that the LuxS protein has an important metabolic function in the recycling of SAH. They also show that Ps. aeruginosa is capable of removing AI-2 activity, implying that this molecule may act as a nutrient. In many bacteria AI-2 may in fact represent not a signal molecule but a metabolite which is released early and metabolized in the later stages of growth.