Escherichia coli holoenzyme RNA polymerase is composed of a core enzyme (E, 1 subunit composition ␣ 2␤␤ Ј) associated with one of seven sigma ()-factors that program the complex to engage and initiate transcription at different sets of promoters (1). Thus, the levels and binding properties of alternative -subunits together with factors that modulate their ability to associate with core RNA polymerase are critical for the relative composition of the multiple holoenzymes available for transcription of the distinct promoter classes within the prokaryotic genome. The seven different -factors of E. coli fall into two groups. The larger of these comprises six factors that share notable sequence and functional similarities to the major D

SummaryTranscription from the Pseudomonas-derived 54 -dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the ␤, ␤Ј and 70 subunits of RNA polymerase, which confer prototrophy on ppGpp 0 E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the 54 -RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from 54 promoters.

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two-to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.

Transcription from Pseudomonas-derived ؊24, ؊12 Po promoter of the pVI150-encoded dmp operon is mediated by the 54 -dependent DmpR activator in response to the presence of aromatic pathway substrates in the medium. However, global regulatory mechanisms are superimposed on this regulatory system so that the specific response to aromatic effectors is absent in cultures until the stationary phase is reached. Here we genetically dissect the system to show that the growth phase response is faithfully mimicked by a minimal system composed of the dmpR regulatory gene and the Po promoter regulatory region and can be reproduced in heterologous Escherichia coli. Using this system, we show that the growth phase-dependent DmpR-mediated response to aromatic compounds is limited to fast-growing cultures. Thus, during exponential growth of cultures in minimal media containing different carbon sources, the response to aromatics is immediate, while the response is suppressed in cultures grown on rich media until the exponential-to-stationary phase transition. Elements known to be involved in the DmpR-mediated transcription from Po were analyzed for the ability to influence the growth phase response. Most dramatically, overexpression of DmpR was shown to completely abolish the growth phase response, suggesting that a negatively acting factor may mediate this level of regulation. The possible mechanism of action and integration of the specific regulation of the dmp operonencoded catabolic enzymes with the physiological status of the bacteria are discussed.Pseudomonas sp. strain CF600 harboring the IncP-2 catabolic megaplasmid pVI150 can efficiently grow on phenol, monomethylated phenols, and 3,4-dimethylphenol as sole sources of carbon and energy (40). The 15 structural genes for the enzymes of the catabolic pathway are located within the plasmid-encoded dmp operon (Fig. 1A) (43). Complete mineralization of pathway substrates is achieved by hydroxylation of the phenolic ring to form catechol, followed by a conversion to Krebs cycle intermediates pyruvate and acetyl coenzyme A via the sequential steps of the meta-cleavage pathway (reviewed in reference 35). Transcription of the dmp operon from the operon promoter, Po, is tightly regulated by the divergently transcribed dmpR gene product so that the enzymes of the pathway are expressed only in the presence of pathway substrates or structural analogs ( Fig. 1) (33,39,41).DmpR belongs to the prokaryotic enhancer-binding family of 54 -dependent regulators, which function to positively control transcription from Ϫ12, Ϫ24 promoters recognized by RNA polymerase utilizing the alternative sigma factor, 54

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related54 -dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding 54 RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of 54 -dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.