Richard J. Lamont

Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS

ABSTRACT The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis,Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway inVibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phaseA. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS−, a recombinant strain in whichluxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression ofafuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in transcomplemented a luxS knockout mutation in P. gingivalis by modulating the expression of theluxS-regulated genes uvrB andhasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

Cloning and Expression of an Adhesin Antigen of Streptococcus sanguis G9B in Escherichia coli

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.

Bacterial cell-to-cell communication : role in virulence and pathogenesis

Introduction Donald R. Demuth and Richard Lamont 1. Quorum sensing and regulation of P. aeruginosa infections Victoria E. Wagner and Barbara Iglewski 2. The Pseudomonas aeruginosa quinolone signal Everett Pesci 3. Quorum sensing mediated regulation of plant/bacteria interactions and A. tumafaciens virulence Catharine C. White and Stephen C. Winans 4. Jamming bacterial communications: new strategies to combat bacterial infections and the development of biofilms Michael Givskov and Morton Hentzer 5. Quorum sensing-mediated regulation of biofilm growth and virulence of Vibrio cholerae Jun Zhu and John. J. Mekalanos 6. LuxS in cellular metabolism and cell-cell signaling Kangmin Duan and Michael Surette 7. LuxS dependent regulation of E. coli virulence Marcie B. Clarke and Vanessa Sperandio 8. Quorum sensing and cell-to-cell communication in the dental biofilm Donald R. Demuth and Richard Lamont 9. Quorum sensing-dependent regulation of staphylococcal virulence and biofilm development Jeremy Yarwood 10. Cell density dependent regulation of streptococcal competence M. Dilani Senadheera, Celine Levesque and D. Cvitkovitch 11. Signaling by a cell surface-associated signal during fruiting body morphogenesis in Myxococcus xanthus Lotte Sogaard-Andersen.

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.

Quorum-sensing-mediated regulation of plant–bacteria interactions and Agrobacterium tumefaciens virulence

INTRODUCTION Plant-associated bacteria have a wide range of interactions with their hosts, from non-specific associations to more dedicated symbiotic or pathogenic interactions. Many complex interactions take place between plant roots and associated bacteria, fungi, and protozoa in a highly diverse and dense community within the rhizosphere. Bacterial cell-to-cell communication systems in this ecological niche appear to affect biofilm formation, pathogenesis, and production of siderophores and antibiotics. These activities are no doubt important in root colonization as well as in symbiosis and pathogenesis. Exciting developments and current studies in understanding the many complex interactions in the rhizosphere include both the characterization of the microbial communities involved and the responses of the plant hosts to these communities. Cell-to-cell signaling between members of the community is no doubt critical for these interactions to sense population densities and diffusion barriers in the rhizosphere. Such studies are beyond the scope of this chapter, but we refer the reader to recent reviews of this field (43, 65, 82). Perhaps the best-characterized group of soil bacteria that serves as the model for understanding plant–bacteria associations is the Rhizobiaceae. This family, in the alpha subgroup of the Proteobacteria, includes members of the genera Rhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium , and Bradyrhizobium (collectively referred to here as rhizobia), which form symbiotic relationships with host plants, and several pathogenic species of the genus Agrobacterium (including A. tumefaciens, A. rhizogenes, A. vitis , and A. rubi , here referred to as agrobacteria).