In addition to producing lethal antibiotics, microorganisms may also use a new form of antagonistic mechanism in which signal molecules are exported to influence the gene expression and hence the ecological competence of their competitors. We report here the isolation and characterization of a novel signaling molecule, cis-2-dodecenoic acid (BDSF), from Burkholderia cenocepacia. BDSF is structurally similar to the diffusible signal factor (DSF) that is produced by the RpfF enzyme of Xanthomonas campestris. Deletion analysis demonstrated that Bcam0581, which encodes an RpfF homologue, was essential for BDSF production. The gene is highly conserved and widespread in the Burkholderia cepacia complex. Exogenous addition of BDSF restored the biofilm and extracellular polysaccharide production phenotypes of Xanthomonas campestris pv. campestris DSF-deficient mutants, highlighting its potential role in inter-species signaling. Further analyses showed that Candida albicans germ tube formation was strongly inhibited by either coculture with B. cenocepacia or by exogenous addition of physiological relevant levels of BDSF, whereas deletion of Bcam0581 abrogated the inhibitory ability of the bacterial pathogen. As B. cenocepacia and C. albicans are frequently encountered human pathogens, identification of the BDSF signal and its activity thus provides a new insight into the molecular grounds of their antagonistic interactions whose importance to microbial ecology and pathogenesis is now becoming evident.

Obligately aerobic tubercle bacilli are capable of adapting to survive hypoxia by developing into a nonreplicating or dormant form. Dormant bacilli maintain viability for extended periods. Furthermore, they are resistant to antimycobacterials, and hence, dormancy might play a role in the persistence of tuberculosis infection despite prolonged chemotherapy. Previously, we have grown dormant Mycobacterium bovis BCG in an oxygen-limited Wayne culture system and subjected the bacilli to proteome analysis. This work revealed the upregulation of the response regulator Rv3133c and three other polypeptides (␣-crystallin and two "conserved hypothetical" proteins) upon entry into dormancy. Here, we replaced the coding sequence of the response regulator with a kanamycin resistance cassette and demonstrated that the loss-of-function mutant died after oxygen starvation-induced termination of growth. Thus, the disruption of this dormancy-induced transcription factor resulted in loss of the ability of BCG to adapt to survival of hypoxia. Two-dimensional gel electrophoresis of protein extracts from the gene-disrupted strain showed that the genetic loss of the response regulator caused loss of the induction of the other three dormancy proteins. Thus, the upregulation of these dormancy proteins requires the response regulator. Based on these two functions, dormancy survival and regulation, we named the Rv3133c gene dosR for dormancy survival regulator. Our results provide conclusive evidence that DosR is a key regulator in the oxygen starvation-induced mycobacterial dormancy response.

SummaryXanthomonas campestris pv. campestris (Xcc) is known to regulate virulence through a quorumsensing mechanism. Detection of the quorumsensing signal DSF by sensor RpfC leads to activation of the response regulator RpfG, which influences virulence by degrading cyclic-di-GMP and by subsequent increasing expression of the global regulator Clp. In this study, we show that mutation of a response regulator RavR containing the GGDEF-EAL domains decreases Xcc virulence factor production. The functionality of RavR is dependent on its EAL domain-associated cyclic-di-GMP phosphodiesterase activity. Deletion of a multidomain sensor gene ravS, which shares the same operon with ravR, results in similar phenotype changes as the ravR mutant. In addition, the sensor mutant phenotypes can be rescued by in trans expression of the response regulator, supporting the notion that RavS and RavR constitute a two-component regulatory system. Significantly, mutation of either the PAS domain or key residues of RavS implicated in sensing low-oxygen tension abrogates the sensor activity in virulence regulation. Moreover, similar to the DSF signalling system, RavS/RavR regulates virulence gene expression through the global regulator Clp. These results outline a co-regulation mechanism that allows Xcc to integrate population density and environmental cues to modulate virulence factor production and adaptation.

Oxygen starvation triggers the shiftdown of the obligate aerobe Mycobacterium bovis BCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the ␣-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.Mycobacteria are obligate aerobes; i.e., they require oxygen for growth. However, it has been known for years that tubercle bacilli encounter hypoxic environments in acute disease, as well as in latent infection (32,34,39,40,45), and the capability of tubercle bacilli to adapt to hypoxic conditions appears to play a role in vivo (3,29,49). Wayne established a link between oxygen starvation and drug resistance. That author demonstrated that upon depletion of oxygen in culture, the bacillus terminates growth and develops into a defined dormant form (41)(42)(43)47). Importantly, the dormant form of the bacterium was found to be resistant against conventional antimycobacterials (46, 47). Hence, hypoxic dormant bacteria could, at least in part, be responsible for the observed persistence of the pathogen during chemotherapy (11).To study the dormancy response in tubercle bacilli, we applied the dormancy culture system that was developed by Wayne for Mycobacterium tuberculosis (47) to the attenuated BCG Pasteur ATCC 35734 strain of M. bovis (19,20,24,31). Wayne's dormancy culture system is based on growth of the bacilli under oxygen-limited conditions in sealed tubes with stirring. Initially, the cultures grow exponentially and consume oxygen rapidly. A temporal oxygen gradient is generated, and the cultures terminate growth when the oxygen concentration reaches a hypoxic threshold level. The bacilli in the hypoxic stationary phase are in a reversible, apparently diploid, synchronized state of low metabolic activity, in which the cells maintain viability for extended periods without division (24, 47); i.e., the organisms are in a physiological state of dormancy, as defined by Kaprelyants et al. (22).Our knowledge of the molecules involved in the mycobacterial dormancy response is fragmentary (5,15,17,18,26,30,37). Elegant biochemical work showed that dormant bacilli adapt their metabolism to anaerobiosis by switching to nitrate respiration (48) and reductive amination of glyoxylate (44). Genetic analysis demonstrated that the stringent response plays a crucial role in the adaptation to hypoxic stationaryphase survival (33). However, only a single polypeptide, the ␣-crystallin homolog, has been shown to be up-regulated in oxygen-starved tubercle bacilli (7, 38, 51, 52). The induction of this chaperon appears to be under control of the sigma factor SigF (4, 9, 10, 25). In this study, the dormancy response in BCG was analyzed using two-dimensional gel electrophoresis (21) to identify new dormancy-induced proteins.Detection and identification of dormancy-induced proteins. Figure 1 (solid circles) shows the growth curve of BCG in the Wayne dormancy culture system. Bacilli were harvested at various time ...

With 2 million deaths per year, TB remains the most significant bacterial killer. The long duration of chemotherapy and the large pool of latently infected people represent challenges in disease control. To develop drugs that effectively eradicate latent infection and shorten treatment duration, the pathophysiology of the causative agent Mycobacterium tuberculosis needs to be understood. The discovery that the tubercle bacillus can develop a drug-tolerant dormant form and the identification of the underlying genetic program 10 years ago paved the way for a deeper understanding of the life of the parasite inside human lesions and for new approaches to antimycobacterial drug discovery. Here, we summarize what we have learnt since the discovery of the master regulator of dormancy, DosR, and the key gaps in our knowledge that remain. Furthermore, we discuss a possible wider clinical relevance of DosR for ‘nontuberculous mycobacteria’.