Cloning and Analysis of the rnc-era-recO Operon from Pseudomonas aeruginosa

Abstract

The rnc operon from Pseudomonas aeruginosa has been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of era mutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.

The rnc operon of Escherichia coli contains three genes, called rnc (6, 11), era (4), and recO (9), that are important for RNA metabolism, cell cycle control, and DNA recombination, respectively. Homologs of each of these genes have been identified in other eubacteria (13), but their functions are best understood in E. coli. The RNase III protein, coded by the rnc gene, is a double-stranded endoribonuclease that assists in the maturation of stable RNAs such as the 16S and 23S rRNAs. It also cleaves mRNA and, in this regard, plays a role in global gene control (6). The Era protein is a GTPase that is required for the growth of bacterial cells (8, 11). Era affects the E. coli cell cycle through its GTPase activity (4) and may function as a gatekeeper by controlling entry of the cell into cytokinesis (4). Recent evidence suggests that Era binds RNA (4a, 10b). The RecO protein participates in DNA recombination and repair. Of these three gene products, Era appears to play a unique and critical role in the survival of the bacterial cell.

The structure of the rnc operon is conserved among E. coli, Salmonella typhimurium, Haemophilus influenzae, and Coxiella burnetii (2, 13, 14). In E. coli, translation of the second gene, era, appears to be coupled to that of the first, rnc, and expression is maintained at a low level, due in part to autogenous regulation of the operon transcript by RNase III (3). The translation of both rnc and era is regulated by the growth rate (4). This rnc operon structure is not conserved among all bacteria in which homologous genes for rnc and era have been identified (13), and this kind of coordinated expression may not be characteristic of all eubacteria. Nevertheless, there is growing evidence that RNase III and Era may share a common function in the maturation of rRNA and the assembly of the ribosome (10). The continued discovery and characterization of similar genes in other organisms, including eukaryotes, promise to yield a more complete understanding of Era’s structure and function (4). Era is of particular interest because it represents a new class of regulator for growth and cell division, and it is the least understood of the three genes in the rnc operon (4). We report here the characterization of the era gene and the rnc operon of Pseudomonas aeruginosa.

The rnc operon of P. aeruginosa was isolated on a clone from a plasmid library of genomic DNA by functional complementation of an era null mutant in E. coli. The genetic system used is based on the induction and curing of a lysogenic lambda prophage carrying the only cellular copy of era, thus generating a cell lacking an era gene. This complementation system, which will be described in detail separately, has proven useful for the selection in E. coli of random and engineered era mutations, as well as homologous era genes from other organisms (10c). Here it was used to screen a pUC18-derived plasmid library made by partial digestion of genomic DNA from P. aeruginosa PAK (a gift from S. Lory). Three plasmids capable of complementing the loss of era were selected from this P. aeruginosa library. One was chosen for further study and labeled pAK-2. A 2.4-kb EcoRI fragment was subcloned from pAK-2 into the vector pBlueScript SK(−) (Stratagene) to make plasmid pBP110. The ability of pBP110 to complement era mutants in E. coli was confirmed, and then it and pAK-2 were used for sequencing and further genetic analyses.

Both strands of the insert DNA contained in plasmids pAK-2 and pBP110 were sequenced with the ABI Prism DNA Sequencing kit and the model 373A DNA sequencer (both from Perkin-Elmer Applied Biosystems). Nucleotide sequences were assembled with Sequencher 3.0 (Gene Codes Corp.) and analyzed with the Genetics Computer Group (Madison, Wis.) package, version 8.0, and the BLAST family of algorithms accessible on the National Center for Biotechnology Information website (10a). The cloned DNA was found to contain genes for rnc and recO as well as a complete era homolog. The respective RNase III and Era proteins encoded by P. aeruginosa and E. coli are nearly 60% identical in amino acid sequence. As is found in the other rnc operons, the stop codon of rnc overlaps the start codon of era. The DNA sequence from the clone agrees almost completely with that now contained in the database set forth by the (as yet incomplete) P. aeruginosa genome project (10d). The similarity in primary structure of this rnc operon to those of E. coli and S. typhimurium supports the idea that transcriptional and translational coupling (2, 3) may also occur within the rnc operon of P. aeruginosa. The rnc-era-recO operon structure appears to be conserved in several gram-negative bacteria (13). This concurs with the conventional belief about the evolutionary relatedness of this group of eubacteria.

The predicted functions of these genes were tested by complementation mapping in E. coli using the original pAK-2 plasmid and its derivatives. Plasmids carrying the three genes were able to substitute and complement mutations of their respective homologous genes in the E. coli host (data not shown). In addition to the original test of era function by selecting for the loss of native era, the P. aeruginosa era also complemented two other conditionally defective era mutants containing the mutations rnc40 (11) and era(Ts) (7). Importantly, plasmids that altered the integrity or expression of the P. aeruginosa era gene were unable to complement era mutants of E. coli (Fig. 1). Thus, by functional complementation and structural similarity to homologous genes, the genes cloned on pAK-2 and its derivatives are proposed to comprise the rnc-era-recO operon of P. aeruginosa.

FIG. 1.

Physical and genetic map of the rnc-era-recO region from P. aeruginosa. A plasmid containing the 2.4-kb rnc operon and its derivatives are shown under the control of the lac promoter (arrowheads). Shaded arrows depict the extent of genes present on each plasmid, with truncations indicated by shorter arrows. These plasmids were introduced into E. coli BSP123 [recA1 srl::Tn10 araD139 Δ(ara-leu)7697 Δ(lac)X74 galU galK rpsL hsdR Δ(rnc-era)::Km^r containing prophage λcI857 Δ(exo-bet-gam) rnc⁺ era⁺ recO′::lacZYA] and analyzed for their ability to complement the loss of era as measured by the efficiency of plating (E.O.P.) following curing of the prophage. Briefly, cells were grown to logarithmic phase, and the prophage was induced at 42°C for 5 min; cells were returned to 30°C and then allowed to recover overnight at 30°C to allow prophage segregation and curing. Approximately 70 to 80% of the cells are cured of the prophage by this technique (12). Cells were then plated at either 30 or 42°C. Uncured cells containing the prophage are killed at 42°C by the induced phage. Thus, only cured cells containing a functional Era survive at 42°C. The E.O.P. was calculated as the number of viable cells at 42°C divided by the viable cell count at 30°C. Note that the E.O.P. shown is the average from at least four independent experiments.

Chopade et al. (5) purified and characterized a membrane-associated protein of 48 kDa that possesses intrinsic GTPase activity and could be autophosphorylated in vitro, properties related to those of Era. Furthermore, polyclonal antibodies against the Era of E. coli recognized the newly discovered protein, and the sequence of its 14 N-terminal amino acids was found to be most similar to the N-terminal sequences of Era proteins from E. coli, S. typhimurium, and H. influenzae (see Fig. 1B in reference 5). For these and other reasons, it was proposed to be the Era equivalent in P. aeruginosa and was called Pra (6). However, a coding sequence for the N-terminal peptide region of Pra is not yet present in the P. aeruginosa genome project database. Moreover, the putative N-terminal 14-amino-acid sequence encoded by the era-complementing gene described here is unlike that of Pra. Thus, in the absence of a gene for Pra, its identity remains in question. The following reasoning may be helpful in understanding why Pra might have been confused for Era. First, the observed similarity of the N-terminal 14-amino-acid sequence of Pra to those of some Era proteins may be strongly influenced by the presence of a common N-terminal GTPase structural domain. In fact, sequences that specifically distinguish Era from other types of GTPases lie beyond the GTPase domain in the C-terminal end of Era (13). Further comparisons await more C-terminal peptide sequence from Pra or the definition of a sequenced gene that matches Pra. Second, at 48 kDa, Pra is ∼30% larger than other bacterial Era proteins. The presumptive Era protein encoded by the clone described here is 34.5 kDa, which is very similar to the 33.8-kDa Era from E. coli. Third, since the GTPase domains of several GTPase protein families are structurally similar, they may present cross-reactive epitopes during antibody selection. This could explain how polyclonal antibodies raised against Pra or Era recognize and bind the GTPase domains of nonhomologous GTP binding proteins. Another polyclonal antibody generated against a C-terminal peptide of E. coli Era recognizes both the E. coli and the P. aeruginosa Era (Fig. 2). As predicted, the P. aeruginosa Era is slightly larger than the Era of E. coli. A similar-sized band was also detected in whole-cell extracts of PAK (data not shown).

FIG. 2.

Western blot analysis of P. aeruginosa Era expressed in E. coli. Cells were grown to logarithmic phase, and the cell equivalent of 20 μg of total protein was loaded in each lane of a sodium dodecyl sulfate–12.5% polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane. The polyclonal antibody used to detect Era is directed against the C-terminal peptide (SDDERALRSLGYGDL) of E. coli, which is very similar to the corresponding region in the P. aeruginosa Era protein (ADDERALRSLGYVDDL). The secondary antibody was a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G, and reactive bands were detected by chemiluminescence. Lane A, W3110 alone; lane B, W3110 containing pHKP5F with the P. aeruginosa era gene (shown in Fig. 1); lane C, BSP123 containing pHKP5F after curing of the prophage and the E. coli era gene (see the Fig. 1 legend).

While the possibility of two different era genes in the P. aeruginosa genome cannot yet be ruled out, there is no example of multiple era genes in the organisms whose genomes have been completely sequenced. In support of this, only clones containing the DNA of the unique era gene, like that described here, complement an era defect in E. coli (Fig. 1) (13, 14). For the reasons stated above, we suggest that Pra is not the Era-equivalent protein of P. aeruginosa. The evidence reported here supports the assignment of P. aeruginosa Era as that GTPase encoded on the pAK-2 plasmid. What gene does Pra represent if it is not Era? One candidate is ThdF, a GTP-binding protein active in thiophene and furan oxidation, whose counterpart in E. coli is of a similar molecular size (48.9 kDa) (1). Unfortunately, no thdF gene homologous to that of E. coli has been defined yet in the P. aeruginosa genome sequence, and thus a definitive assignment of Pra awaits further evidence.

Nucleotide sequence accession number.

The nucleotide sequence of the P. aeruginosa rnc operon has been submitted to GenBank under accession no. AF123492.