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 Sadenosylhomocysteine (SAH) or 5'-methylthioadenosine (MTA) serves as a substrate for AI-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5α (which carries a luxS frame-shift mutation) were capable of generating AI-2 activity upon addition of SAH, but not MTA. S-Ribosylhomocysteine (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.

The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that in Staphylococcus aureus the luxS gene forms a monocistronic transcriptional unit under the control of a 70 -dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation of luxS in various S. aureus strains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and the agr signaling system. Conversely, AI-2 production remained unchanged in an agr mutant. However, luxS mutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture, luxS mutants of various S. aureus strains showed reduced ability to compete for growth under these conditions. In contrast, a complemented luxS mutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness of S. aureus RN6390B during intracellular growth in epithelial cells: the wild type and a luxS mutant showed very similar growth patterns after their internalization by MAC-T cells.Many bacteria, including pathogens and commensals, are known to communicate via diffusible signal molecules (26,63). It is often assumed that these molecules are employed to regulate genes in concert with cell population density (quorum sensing). Bacteria of the genus Staphylococcus are known to possess an autoinducing peptide (AIP)-based signaling system, encoded by the agr locus, the function of which has been studied in detail in Staphylococcus aureus and Staphylococcus epidermidis (for reviews see references 36 and 49). In S. aureus, this system is involved in the regulation of many exoproteins, including exoenzymes, exotoxins, and surface proteins (49). Sequence analysis of completed genomes revealed that Staphylococcus spp., like many other bacteria, also contain a luxS gene and therefore may employ a second signaling system based on the furanone derivative, autoinducer 2 (AI-2).The LuxS/AI-2 system has been analyzed in detail in Vibrio spp., in particular Vibrio harveyi and Vibrio cholerae, where it is involved in the regulation of bioluminescence and virulenceassociated traits, respectively (24,25,34,40) (for a review, see reference 76). Synthesis of AI-2 depends on the enzyme LuxS (55, 71), which generates the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione (DPD) from S-r...

The aim of the present study was to examine the stability and evolution of tet(M)-mediated resistance to tetracyclines among members of different clonal lineages of Streptococcus pneumoniae. Thirty-two tetracyclineresistant isolates representing three national (Spanish serotype 14, Spanish serotype 15, and Polish serotype 23F) and one international (Spanish serotype 23F) multidrug-resistant epidemic clones were all found to be tet(M) positive and tet(O), tet(K), and tet(L) negative. These isolates all carried the integrase gene, int, which is associated with the Tn1545-Tn916 family of conjugative transposons. High-resolution restriction analysis of tet(M) products identified six alleles, tet(M)1 to tet(M)6: tet(M)1 to tet(M)3 and tet(M)5 in isolates of the Spanish serotype 14 clone, tet(M)4 in both the Spanish serotype 15 and 23F clones, and tet(M)6, the most divergent allele, in the Polish 23F clone. This indicates that tet(M) variation can occur at the inter-and intraclone levels in pneumococci. Two alleles of int were identified, with int1 being found in all isolates apart from members of the international Spanish 23F clone, which carried int2. Susceptibility to tetracycline, doxycycline, and minocycline was evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracyclines. Resistance to tetracyclines was found to be inducible in isolates of all clones; however, the strongest induction was observed in the Spanish serotype 15 and 23F clones carrying tet(M)4. Tetracycline was found to be the strongest inducer of resistance, and minocycline was found to be the weakest inducer of resistance.The gram-positive pathogen Streptococcus pneumoniae (the pneumococcus) is a major cause of pneumonia, otitis media, and meningitis (12). The evolution and broad global distribution of multiple antibiotic resistance determinants in bacteria have resulted in a situation in which pneumococci are commonly resistant to penicillin, the broad-spectrum cephalosporins, macrolides, lincosamides, co-trimoxazole, chloramphenicol, and tetracyclines, as well as rifampin (11), sulphonamides (42), and fluoroquinolones (13,24,30), making the treatment of serious pneumococcal disease increasingly difficult (17,22). The transformable nature of S. pneumoniae (which has played an important role, along with point mutations) in the evolution of resistance [1,4,11,13,24,30] has in no small part also led to a population structure characterized by free genetic exchange, punctuated by clonal expansion of successful variants. The best studied of these are the Spanish 23F, Spanish 6B, and French/ Spanish 9V14 multidrug-resistant clones that have now spread intercontinentally (see reference 10 for a recent review).One class of antimicrobial agents found most often in clinical use is the tetracyclines, broad-spectrum bacteriostatic drugs shown to be active against pneumococci (33). In some European (9, 16, 23), Asian (35,36,41,47), and African (31, 52), countries lack of susceptibility to tetracyclines...

The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.