Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly. This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria. Here we review the quorum-sensing circuits of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Vibrio cholerae. We outline these canonical quorum-sensing mechanisms and how each uniquely controls virulence factor production. Additionally, we examine recent efforts to inhibit quorum sensing in these pathogens with the goal of designing novel antimicrobial therapeutics.

Bacteria cycle between periods when they perform individual behaviors and periods when they perform group behaviors. These transitions are controlled by a cell-cell communication process called quorum sensing, in which extracellular signal molecules, called autoinducers (AIs), are released, accumulate, and are synchronously detected by a group of bacteria. AI detection results in community-wide changes in gene expression, enabling bacteria to collectively execute behaviors such as bioluminescence, biofilm formation, and virulence factor production. In this study, we show that the transcription factor AphA is a master regulator of quorum sensing that operates at low cell density (LCD) in Vibrio harveyi and Vibrio cholerae. In contrast, LuxR (V. harveyi)/HapR (V. cholerae) is the master regulator that operates at high cell density (HCD). At LCD, redundant small noncoding RNAs (sRNAs) activate production of AphA, and AphA and the sRNAs repress production of LuxR/HapR. Conversely, at HCD, LuxR/HapR represses aphA. This network architecture ensures maximal AphA production at LCD and maximal LuxR/HapR production at HCD. Microarray analyses reveal that 300 genes are regulated by AphA at LCD in V. harveyi, a subset of which is also controlled by LuxR. We propose that reciprocal gradients of AphA and LuxR/ HapR establish the quorum-sensing LCD and HCD gene expression patterns, respectively.

The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP I , on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP I disrupts interactions with promoter DNA at least 25 Å away, between positions À6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP I . Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.[Keywords: RNA polymerase; promoter; DksA; ppGpp; transcription initiation; ribosome synthesis] Supplemental material is available at http://www.genesdev.org. DksA, ppGpp, and NTPs work together to regulate rRNA synthesis in Escherichia coli (Paul et al. 2004). DksA concentrations are relatively constant (Rutherford et al. 2007), but ppGpp and NTP concentrations vary dramatically with nutrient availability (Murray et al. 2003). Inactivation of the dksA gene derepresses rRNA transcription, uncoupling ribosome production from the cellular demand for protein synthesis, because direct modification of RNA polymerase (RNAP) by DksA is needed for changes in the concentrations of ppGpp and NTPs to exert effects on the transcription initiation complex (Paul et al. 2004).The mechanism of DksA action remains unclear. Unlike conventional regulators of transcription initiation, DksA does not bind to DNA but instead interacts directly with RNAP (Paul et al. 2004;Perederina et al. 2004). Biochemical studies and structural similarities between DksA and the transcription elongation factors GreA and GreB suggest that DksA binds in the RNAP secondary channel (Opalka et al. 2003;Perederina et al. 2004; S.T. Rutherford, I. Toulokhonov, C.E. Vrentas, W. Ross, and R.L. Gourse, unpubl.), but there is no structure of DksA bound to RNAP, and the precise interactions between RNAP and DksA have yet to be defined.Because DksA binds RNAP instead of a specific DNA sequence, it has the potential to affect all promoter complexes. Consistent with this prediction, DksA decreases the lifetimes of complexes formed by all promoters tested to date (Paul et al. 2004(Paul et al. , 2005Rutherford et al. 2007). However, DksA directly affects transcriptional output only from a subset of promoters, including many needed for the synthesis of ribosomes, virulence, membrane stress responses, and amino aci...

SummaryEscherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfill some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentrations of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When overexpressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA only modestly affected rRNA promoters in vitro, and even when overexpressed GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the ω subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

bBacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes. LuxR strongly controls genes at both low cell density and high cell density, suggesting that it is the major quorum-sensing regulator. In contrast, AphA is absent at high cell density and acts to fine-tune quorum-sensing gene expression at low cell density. We examined two loci as case studies of coregulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum-regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group life-styles. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at midcell density. Thus, the asymmetric production of AphA and LuxR coupled with differences in their strengths and timing of target gene regulation generate a precise temporal pattern of gene expression.