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In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing. Two quorum-sensing regulons have been identified in which the LuxR homologues LasR and RhlR are activated by N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively. The lasR and rhlR genes are linked to the luxl homologues lasl and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRl and rhlRl are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship. By using lacZ transcriptional fusions in both homologous (P. aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhlR expression is regulated by LasR/OdDHL, and (iii) that RhlR/BHL regulates rhll. We also show that expression of the stationary-phase sigma factor gene rpoS is abolished in a P. aeruginosa lasR mutant and in the pleiotropic BHL-negative mutant PANO67. Furthermore, our data reveal that kin E. coli, an rpoS-lacZ fusion is regulated directly by RhlR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum-sensing cascade involving RhlR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.

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Background: All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase a factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (ar3) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (Ea38) was found to recognize in vitro a number of typical a70-type promoters, including the lacUV5 and thp promoters. Some, however, were recognized exclusively or preferentially by Ear70, whereas at least one, fic, was favored by E&rm. Thus E. coli promoters can be classified into three groups: the first group is recognized by Ea70 and Ea,38 but the second and third groups are recognized substantially by either Ea70 or Eam alone. In contrast to other minor afactors, am shares a set of amino acid sequences common among the principal a factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of a38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that am is a second principal a factor in stationary-phase E. coli.The promoter selectivity of RNA polymerase holoenzyme, controlled by replacing cr factors, is the primary mechanism for the global switching of gene expression in eubacteria. The principal Cr factor or70 (the rpoD gene product) in exponentially growing Escherichia coli potentiates the transcription of genes controlled by the consensus promoters consisting of two hexanucleotide sequences, TTGACA and TATAAT (1, 2). Other minor species of alternative cr factors recognize different sets of promoter sequences, each being associated with a limited number of genes that are mostly expressed in response to various stress conditions (1, 2). The rpoS (katF) gene of E. coli was first identified as a positive regulator of the katE gene encoding hydroperoxidase II (HPII) (3). Later, several other genes that are, for the most part, expressed in the stationary growth phase were shown to be regulated by the rpoS allele (4, 5). Thus the rpoS gene was assumed to be the central regulator of gene expression in the stationary phase (4). From the nucleotide sequence ofthe rpoS gene, the gene product has been believed to be an RNA polymerase cr factor (6, 7). However, a, activity has not been detected previously for the rpoS gene product.In this study, we determined the nucleotide sequence ofthe rpoS gene § and found that the gene product is structurally similar to or70 and the principal or factors from divergent eubacterial species (7-12). To assess the functional similarity between the rpoS gene product (or38) and cr70, we reconstituted Eor38 holoenzyme using a highly purified rpoS gene product and examined its promoter selectivity in in vitro transcription assays using various E. coli promoters. MATERIALS AND METHODS Bacterial Strains. E. coli strains DH1 (13), XLlblue (14), MC4100 (4), and BL21(DE3) (15) were used in this study. Plasmid pTZ19R was obtained from Pharmacia. pET3b, pLysS, and pLysE plasmids are as describ...