y of the D-HAI-1 used was confirmed by in vitro phosphorylation experiments with LuxN and LuxU. The L-HAI-1 isomer caused no significant induction, which is in agreement with the known stereospecificity of V. harveyi HAI-1. The dark phenotype of a luxS/cqsA mutant, which produces only HAI-1, is compatible with the low intensity of bioluminescence induced by HAI-1 observed here. By contrast, in the luxM/luxS mutants KM413 and KM135 , bioluminescence could be induced by HAI-1. Autoinducers as Timers It is important to note that all these mutants are able to produce CAI-1. In our experiments bioluminescence was measured of midexponentially grown cells, when CAI-1 was not detectable. In former studies CAI-1 might be responsible for bioluminescence induction, because cells were analyzed after 14 16 h of growth. Note that, as described above, HAI-1 is at no time the sole AI to be found in a wild type culture, and our results indicate that induction of bioluminescence by HAI-1 is dependent on the presence of other AIs. We therefore tested the effects of HAI-1 and AI-2, applied in different molar ratios, on the induction of bioluminescence. Importantly, bioluminescence increased when both HAI-1 and AI-2 were present. This effect was particularly pronounced at the lowest AI-2 concentrations tested and a low concentration of HAI-1; no further increase was observed upon exposure to higher concentrations of HAI-1. Thus, while AI-2 is able to induce bioluminescence in V. harveyi on its own, the simultaneous presence of HAI-1, which has only a minor effect by itself, significantly increases the level of bioluminescence observed. Then we 
tested the dose-dependent effect of AIs on the induction of the exoprotease. An increase 22803826 in the AI-2 concentration led to a concomitant increase in the exoproteolytic activity. HAI-1 induced this activity too, but to a much lesser degree. Finally, a mixture of HAI-1 and AI-2 resulted in maximal exoproteolytic activity. These results correlate with the onset of exoproteolytic activity in a growing wild type population at a time when both HAI-1 and AI-2 are present in the medium. blends of the AIs. As a control, the synthase negative mutant JMH634, which is unable to produce AI-2, HAI-1, and CAI-1, was analyzed at essentially the same stages of growth. Cells were cultivated, RNA was isolated, cDNA was synthesized, and levels of the luxR transcript were determined by qRT-PCR. Changes in luxR mRNA levels relative to the recA transcript were calculated using the CT method. The level of luxR mRNA in the wild type increased with the buildup in AI-2 concentration, and rose further when HAI-1 appeared in the medium. The maximal transcript level was measured at the time when all three AIs were present. The number of transcripts per cell revealed an increase from 0.9, 2.2, 4.2 to 11.0 transcripts per cell from the early exponential to the stationary growth phase. In the mutant JMH634 0.2 luxR transcripts per cell were detectable, indicating that luxR is not completely repressed in the absence of AIs. However, the effects of extremely low concentrations of LuxR on cell physiology are still unknown. The number of LuxR buy Oritavancin (diphosphate) proteins per cell is difficult to deduce from these data, due to the numerous feedback mechanisms. Nevertheless, it is expected that the number of transcripts is reflected in the number of LuxR molecules produced, which in turn is the primary parameter 9874164 that determines the responses of different gene classes. AI-regul
