Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability

Abstract

tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s).

Fig. 1. Sequence alignment of E.coli and N.gonorrhoeae (strain N400) ssrA genes. Boxes indicate positions of sequence identity. Letters denoting amino acid tag sequences are placed either above (for E.coli) or below (for N.gonorrhoeae) the codons of the tagging sequences. The tag sequences of the two ssrA genes differ at only one codon; codon 5 encodes asparagine in ssrA^Ec and threonine in ssrA^Ng.

Fig. 2. Northern blot analysis identifying tmRNAs of E.coli and N.gonorrhoeae. The probe was synthesized using ssrA^Ec as template. Strains examined were: K37, wild-type for ssrA^Ec (lane 1), K8619, isogenic with K37 except that it has the ssrA^Ec::cat allele (lane 3), and variants of K8619 carrying pGCtm (lane 2) or derivatives of pGCtm with ssrA^Ng mutant alleles (lanes 4–6).

Fig. 3. Functional analysis of ssrA^Ng alleles with transposon insertions in and surrounding ssrA^Ng. A library of 13 m-Tncm insertions was created in plasmid pGCtm. The positions (shown by downward pointing arrows) in the GCtm fragment of these insertions were located by DNA sequencing. Insertions were located in three regions: A, upstream of ssrA; B, within ssrA; and C, downstream of ssrA. The effect on tmRNA activity of each insertion was determined by assessing growth of λimmP22hy25 in derivatives of the ssrA^Ec::cat E.coli strain K8619 carrying variants of pGCtm with the respective 13 insertions (row 1); (+) support of phage growth and (–) no support of phage growth. Formation of stable recombinants with mini-transposon inserted ssrA^Ng alleles in N.gonorrhoeae either haploid (row 2) or diploid (row 3) for ssrA^Ng was determined by selecting Cm^r colonies; (+) Cm^r colonies were isolated and (–) Cm^r colonies were not isolated.

Fig. 4. Variants used in determining the properties of gonococcal tmRNA required for N.gonorrhoeae growth. The DD mutation was generated by replacing the two terminal alanine codons with two aspartic acid codons, while the ochre mutation was generated by changing the fourth codon from GAA to the sequence encoding the ochre translation terminator UAA. Both mutants also carry a single base substitution (from AAACTT to AAGCTT) that provides an identifying HindIII site, as indicated by the underlined sequence above the tag sequence. Two changes were made in the sequences encoding the acceptor stem: these result in the change of the G:U base pair required for recognition of alanyl-tRNA synthetase to a U:G base pair. This mutant also carries a single base substitution (from CCTTGG to CGTTGG) that provides an identifying marker by eliminating a StyI site that is only five nucleotides away from the G3 position, indicated by the asterisk.

Fig. 5. Details of the allelic replacement that placed ssrA^Ng mutant alleles on the gonococcal chromosome. Construction of a recombinant chromosomal ssrA^Ng gene with the ochre mutation is used as an example of the procedure. (A) The double crossover recombination events resulting in allelic replacement. Selection is for Cm^r carried by the linked m-Tncm. The m-Tncm will be crossed onto the chromosome without the ochre mutation if the recombination occurs as shown in (i). The ochre mutation (indicated by the asterisk) will be crossed with the m-Tncm onto the chromosome if the recombination occurs as shown in (ii). Arrows indicate the positions of the primers used in (B) and (C). (B) Schematic representation of the PCR products of the allelic replacements diagrammed in (A). The downward pointing arrows indicate the locations of the HindIII sites used in the analysis of the PCR products shown in (C). The location of the ochre codon is shown in (ii). (C) Cleavage of PCR products confirming proper allelic replacement. Cm^r transformant colonies were used as the source of template DNA for the PCRs. PCR products, generated using the primers indicated in (A), were cleaved with HindIII and the products analyzed by electrophoresis in an agarose gel [source of DNA: lane 1, DNA 1 kb ladder (Gibco); lane 2, wild-type allele; lane 3, plasmid with cloned ssrA^Ng/ochre allele; lanes 4 and 5, transformants]. Because an engineered HindIII site was positioned adjacent to the ochre mutation, PCR products from transformants that have an extra HindIII site, as shown in lane 4, are likely to carry the ochre mutation. Therefore, these transformants were chosen for sequence analysis to determine definitively if the transformant had the desired mutation.

Fig. 6. Substitution of the N.gonorrhoeae tmRNA with E.coli tmRNA in N.gonorrhoeae. (A) Insertion of ssrA^Ec in the N.gonorrhoeae genome at the iga locus. PCR fragments containing ssrA^Ec and the ermC gene (encoding resistance to erythromycin) were cloned into the gonococcal non-essential iga gene and the entire fragment was cloned in plasmid pUP6. The resulting plasmid, pIGA::ssrA^Ec, was trans- formed into N400 and erythromycin-resistant (Em^r) transformants were isolated. These Em^r colonies, shown to be heterodiploid for ssrA (ssrA^Ng/ssrA^Ec), were formed by a double crossover within the iga sequences flanking the ssrA^Ec-ermC insert. Strain C102 is one of these heterodiploid derivatives of N400 that was chosen for further study. Two derivatives of GCtm carrying m-Tncm insertions, B12 (in ssrA^Ng) and C12 (downstream of ssrA^Ng), were used to transform C102 to determine if E.coli tmRNA is sufficient to support gonococcal viability. (B) Northern blot analysis to identify E.coli tmRNA in N.gonorrhoeae. RNA was isolated from bacterial strains whose genotypes are listed at the top of each lane. Two probes were used, one specific for E.coli tmRNA and the other specific for 16S rRNA; the latter served as a control to indicate the relative amounts of RNA loaded in each lane.