Phylogenetic analysis of L4-mediated autogenous control of the S10 ribosomal protein operon

Abstract

We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli. In E. coli, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E. coli leader, particularly in the region which is critical for L4-mediated autogenous control in E. coli. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E. coli. Our results suggest that an E. coli-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E. coli. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.

FIG. 1

Maps of the S10 operon and plasmids used for regulatory studies. (A) Organization of genes in the E. coli S10 operon. (B) Region of the S10 operon amplified by PCR. The site of L4-mediated termination (att) is indicated by the arrowhead. Positions of the primers L1 and L2 are indicated by hatched bars. Open boxes on the primer bars indicate recognition sites for EcoRI (E) and HindIII (H) introduced during the amplification reaction. (C and D). General structures of L4 target and L4 source plasmids. Leader-S10′/lacZ′ or leader-lacZ genes driven by E. coli P_S10 were introduced into cells carrying an IPTG-inducible P_lac-L4 plasmid (C). Leader-S10′/lacZ′ or leader-lacZ genes driven by the IPTG-inducible P_trc were introduced into cells carrying an arabinose-inducible P_ara-L4 plasmid (D). SD-S10 and SD-lacZ refer to the Shine-Dalgarno sequences for the S10 and lacZ structural genes, respectively.

FIG. 2

Alignment of S10 leader sequences. Bases differing from the E. coli sequence are indicated by white text on black background. The approximate positions of presumed hairpins are also shown. Ecoli, E. coli; Styph, S. typhimurium; Cfr, C. freundii; Yentero, Y. enterocolitica; Ypestis, Y. pestis; Smarc, S. marcescens; Mmorg, M. morganii; Hinf, H. influenzae; Vibrio, V. cholerae; Pseudo, P. aeruginosa. Except for the Pseudomonas sequence (see below), the 5′ ends of the various leaders were presumed from potential promoter sequences. The Vibrio leader probably begins about 80 bases upstream of the indicated sequence. The alignment programs did not identify any significant sequence homology in the Vibrio leader upstream of nucleotide 149, and so only the 3′ two-thirds of the leader sequence is shown. The complete sequence is shown in Fig. 3H. The asterisks below the Pseudomonas sequence refer to likely −35 and −10 sequences for the S10 promoter in this species. Therefore, the leader is probably much shorter than the leaders of other eubacteria. However, for comparison the sequence upstream of the presumptive transcription start site is included in the alignment.

FIG. 3

Secondary structure predictions for the S10 leader regions. The secondary structures were predicted by using mfold (55). The computer-predicted E. coli structure has been confirmed by in vitro structure probing analysis (37). The site of L4-mediated transcription termination in the E. coli leader is indicated. Nucleotides differing from the E. coli sequence in the alignments shown in Fig. 2 are indicated by the black circles with white text. The Shine-Dalgarno sequence and the AUG initiation codon for the S10 structural gene are indicated by boxes. entero, enterobacterium.

FIG. 4

Effect of L4 on in vivo synthesis of S10′/β-Gal′ from plasmids carrying foreign S10 leaders. Cells carrying a P_ara-L4 plasmid and a P_trc-leader-S10′/lacZ′ fusion plasmid (Fig. 1D) with the indicated heterologous leader sequence were grown exponentially. Aliquots of the culture were labeled for 2 min with [³⁵S]-methionine immediately before or 20 min after the addition of IPTG to induce expression of S10′/β-Gal′. Twenty-three minutes after IPTG addition, arabinose (ara) was added to the culture to induce L4 oversynthesis; after another 15 min, an aliquot was labeled with [³⁵S]methionine. Total protein extracts were fractionated by PAGE and analyzed by autoradiography. The protein band corresponding to S10′/β-Gal′ is indicated by the horizontal arrows. Control experiments showed that the synthesis of this protein is dependent on the presence of a plasmid carrying the P_trc-leader-S10′/lacZ′ construct, and its regulation is dependent on the leader and on the induction of an active L4 protein (references , , , and and data not shown). A second band, smaller than the S10′/β-Gal′ band, is also induced by IPTG but is not affected by L4 induction. This protein (●) is the product of the lacZ^ΔM15 gene carried by the F′ lac plasmid carried in LL308 (23). It is well resolved in the gels shown in panels A to C but coelectrophoreses with another band in panel D. Ecoli, E. coli; Mmorg, M. morganii; Hinf, H. influenzae; Vibrio, V. cholerae; Pseudo, P. aeruginosa.

FIG. 5

Effect of L4 on in vivo synthesis of β-Gal from plasmids carrying foreign S10 leaders. Cells carrying a P_ara-L4 plasmid and a P_trc-leader-lacZ fusion plasmid (Fig. 1D) with the indicated heterologous leader sequence were labeled as described in the legend to Fig. 4. The protein band corresponding to β-Gal is indicated by the horizontal arrows. The smaller band that is induced by IPTG (●) is the product of the F′ lacZ^ΔM15 gene. Mmorg, M. morganii; Hinf, H. influenzae; ara, arabinose.

FIG. 6

In vitro transcription of E. coli and M. morganii leaders. (A) The general structure of the DNA template, pLL226 or its heterologous derivatives, is shown. The position of the substituted DNA from M. morganii is indicated by the solid bar. P_S10, E. coli promoter for the S10 operon; att, site of L4-stimulated transcription termination; t_rrnC, terminator from rRNA rrnC operon. (B) Transcription reactions were performed in the presence of NusA, and, where indicated, L4. Aliquots were removed at the indicated times after the start of transcription, fractionated on an 8% urea-polyacrylamide gel, and analyzed by autoradiography. Relevant portions of the gel are shown. The RT band contains readthrough transcripts terminated at t_rrnC; the ATT bands contain attenuated transcripts reflecting RNA polymerases paused or terminated at the attenuation site. Ecoli, E. coli; Mmorg, M. morganii.

FIG. 7

Autogenous regulation by L4 in S. typhimurium. S. typhimurium (Styph) or E. coli (Ecoli) cells carrying the indicated plasmids were pulse-labeled with [³⁵S]methionine before (−) or 10 min after (+) addition of IPTG to induce oversynthesis of E. coli L4. See Materials and Methods for details. Total protein extracts were fractionated by PAGE and analyzed by autoradiography. The protein band corresponding to S10′/β-Gal′ (A) or β-Gal (B) is indicated by the horizontal arrows. The band just below the β-Gal band in the E. coli lanes (B) is the product of the F′ lacZ^ΔM15 gene. In panel A, “target” refers to the absence (−) or presence (+) of the P_S10-E. coli S10 leader-S10′/lacZ′ target plasmid.

FIG. 8

Phylogenetic tree of relevant proteobacteria based on 16S rRNA sequences.