The nitrous oxide reductase of Neisseria gonorrhoeae is a transcriptionally active yet translationally silent potogene

Abstract

Many parasitic organisms undergo reductive evolution, the process of losing genes which are now redundant since nutrients can be acquired from the host (Gómez-Valero et al., 2007). Such shortening of the genome may confer a selective advantage upon cell division (D’Souza et al., 2014). The bacterium Neisseria gonorrhoeae is an example of a niche specialist. This obligate human pathogen survives in the urogenital tract, a microaerobic environment, via denitrification. During denitrification, nitrogen oxides (NOX) can be reduced from nitrate (NO3-) to dinitrogen (N2) by a series of metalloenzymes as the terminal electron acceptors in oxidative phosphorylation instead of O2 (Barth et al., 2009a).
N. gonorrhoeae acquires both NOx and the metal ion cofactors needed for denitrification from the host. The key metal is copper (Cu), which is leached from intrauterine contraceptive devices used by a high percentage of women (Crandell and Mohler, 2021). In rare instances when an immune response to N. gonorrhoeae is induced, host phagosomal Cu level is elevated (Djoko et al., 2015), yet gonococci cells survive (Château and Seifert, 2016). Whilst expressing several respiratory cuproenzymes needed for growth and surviving high Cu level, N. gonorrhoeae lacks proteins from the Cue, Cso, Cus and Cop systems for Cu sensing and export (Andrei et al., 2020). Little is known generally about how bacteria acquire and insert Cu into cuproenzymes (Stewart et al., 2019).
N. gonorrhoeae possesses a truncated denitrification pathway; the nitrate reductase (nar/nap) operon is absent and the nitrous oxide reductase (nos) operon is nonsense-mutated. The nitrous oxide reductase enzyme, NosZ, catalyses reduction of nitrous oxide (N2O) to dinitrogen (N2). This reaction is the last in the denitrification pathway and is catalysed by a CuZ site. The nos operon consists of six genes (nosRZDFYL). There is a single nonsense substitution in each of the following gonococcal genes: nosR (the putative transcription regulator and electron donor to NosZ), nosZ (the reductase enzyme) and nosD (part of the Cu delivery system to NosZ). These mutations are unique to N. gonorrhoeae among Neisseria, suggesting they evolved after speciation from N. meningitidis. In NosZ, the nonsense-truncated protein would lack the C-terminal CuA site implicated in electron transfer to CuZ. Hence, the gonococcal nosZ has thus long been assumed to be a non-functional “pseudo” gene (Barth et al., 2009a; Overton et al., 2006).
The premature stop codons in gonococcal nos are conserved across different strains. It is unclear how N. gonorrhoeae may benefit from inactivation of NosZ, particularly in a microaerobic Cu-rich environment (such as that generated by a Cu contraceptive). Hence, we were interested in the possibility that stop codon suppression may be occurring in the gonococcal nos mRNA. In recent decades, much work has been done on how premature stop codons can be readthrough (Feng et al., 2022), and how supposed pseudogenes possess functionality (Li et al., 2013). This has led to authors coining the terms pseudo-pseudogene for putative pseudogenes which are actually functional (Prieto-Godino et al., 2016) and potogene for DNA sequences which have potential to evolve into novel genes (Brosius and Gould, 1992).
Under low Cu levels, the Paracoccus denitrificans NosZ polypeptide is subject to degradation such that a truncated NosZ lacking the C-terminal CuA domain accumulates in the periplasm which still retains activity (Felgate et al., 2012). While the product of gonococcal denitrification has been determined to be N2O, not N2 (Lissenden et al., 2000), the presence of a NosZ protein in N. gonorrhoeae has yet to be experimentally tested.
In this study, we investigated the expression of the gonococcal nos operon and NosZ protein. Our research shows the gonococcal nos operon is fully transcribed and upregulated by NO2-, but a ∆nosZ strain has no distinct phenotype versus the WT. This corroborates other studies which found transcriptionally active but phenotypically silent gonococcal genes (Takahashi and Watanabe, 2005). Expression of the gonococcal nos operon was not found to be influenced by Cu, contradicting findings from other bacterial species that Cu upregulates nos expression (Felgate et al., 2012; Sullivan et al., 2013; Woolfenden et al., 2013). Despite transcription, we have determined the NosZ protein is not stably expressed. Supplementation of 1 and 10 µM Cu in N. gonorrhoeae cultures does not generate a detectable NosZ protein. We suggest this lack of a stable gonococcal NosZ protein may be due to nonsense mutations in the nosRZD genes causing degradation of the nascent polypeptide, or inhibition of translation by mRNA secondary structures such as hairpins.
Other Neisseria thought to lack NosZ include N. meningitidis, another obligate human pathogen (Stephens, 2009) and N. mucosa, an emerging opportunistic human pathogen (Osses et al., 2017). N. meningitidis has a deletion from the middle of nosR to the middle of nosD, eliminating the catalytic subunit nosZ, in addition to premature stop codons in nosY and nosX, while N. mucosa contains a premature stop codon within nosZ and a deletion in nosF (Barth et al., 2009a). Commensal Neisseria species largely retain a functioning denitrification pathway. We propose the pathogenic Neisseria independently lost the requirement for N2O reduction by either acquiring some yet unknown alternative respiratory mechanism, or compensation by the host minimising the need for the last step of denitrification. Alternatively, the nos mRNA transcripts in these species may have an unknown regulatory role which provides a selective advantage (Kamieniarz-Gdula and Proudfoot, 2019).
We hope these findings may contribute to an increased understanding of potogenes, as well as the evolution and pathogenicity of Neisseria species. Determining the evolutionary drivers behind this loss of protein expression in pathogenic Neisseria species is an objective for future research.