Wade C. Winkler

Bacterial transcription elongation has been investigated most thoroughly for the model bacterium Escherichia coli. The E. coli elongation factor NusG associates through its N-terminal domain with the transcription elongation complex (TEC), while its C-terminal domain forms mutually exclusive interactions with either ribosomal protein S10 or Rho termination factor. The E. coli NusG:S10 complex bridges the TEC with a pioneering ribosome for coupling of transcription and translation. NusG also participates in antitermination complexes (ATC) that inhibit Rho-dependent termination and increase transcription of rRNA operons. A characteristic sequence in the nascent rrn RNA, called boxA , loads E. coli NusB:S10 onto the NusG-bound TEC, leading to the formation of the rrn ATC. However, these findings may not extend to all bacteria. Rho-dependent termination is less important in gram-positive bacteria, including some that do not encode Rho, and transcription and translation remain uncoupled in Bacillus subtilis and many other gram-positive bacteria. It is therefore unclear whether the interaction network for E. coli elongation factors is conserved in these organisms. We identified boxA -like sequences upstream of 16S and 23S genes in Bacillus species and found that they associate directly with NusB. We also found that B. subtilis S10 associates with both NusB and NusG, and that Rho can also associate with NusG. Bioinformatic analyses revealed conservation of interaction interface residues across multiple phyla. Together, these data demonstrate that the core interaction network of bacterial elongation factors is broadly conserved across diverse bacteria, despite differences in how these interactions function in individual species. IMPORTANCE: Escherichia coli is known to use several key elongation factors that promote direct coupling of transcription with translation. These factors also trigger the formation of an antitermination complex that protects the synthesis of ribosomal RNA and CRISPR transcripts from interference by the Rho termination factor. However, Bacillus subtilis is not thought to couple transcription with translation. It is also not known whether B. subtilis needs to suppress Rho, given that its Rho is dispensable. Therefore, we purified B. subtilis elongation factors and mapped their interaction network. Surprisingly, we find that the B. subtilis elongation factors form complexes that closely resemble E. coli counterparts, despite the significant mechanistic differences in how these bacteria control transcription elongation.

The transcription factor NusG is essential for the bacterial transcription elongation complex (TEC). During elongation, NusG dynamically interacts with other proteins, affecting transcription-translation coupling, processivity, and termination. NusG is integral to antitermination complexes, where it associates with other factors to modify the TEC, allowing it to bypass termination sites. In Gammaproteobacteria, these complexes enhance the expression of ribosomal RNA operons, bacteriophage operons, and CRISPR arrays. Specialized NusG paralogs, such as LoaP, RfaH, and UpxY, are believed to form their own unique antitermination complexes. RfaH, the only well-studied paralog, binds to a paused TEC via a specific nontemplate DNA sequence. The RfaH-bound TEC then enhances transcription-translation coupling while also preventing Rho termination. However, it is unclear whether the other NusG paralogs use similar mechanisms, nor have antitermination complexes been investigated for Gram-positive bacteria. Previously, Bacillus velezensis LoaP was shown to enhance transcription of the difficidin antibiotic synthesis operon, but its antitermination determinants were unknown. In this study, we purified RNA polymerase and transcription factors and reconstituted difficidin operon ( dfn ) antitermination activity in vitro . The dfn 5′ leader region contains a small RNA hairpin essential for antitermination in vivo and in vitro , and an intrinsic terminator that is highly dependent on NusA. The antitermination activity of LoaP emanates from its ability to specifically antagonize NusA; no other factors are required. These findings demonstrate the basic requirements of the LoaP class of NusG paralogs and indicate that the LoaP antitermination mechanism differs significantly from that of the RfaH paradigm. IMPORTANCE: Genes encoding the NusG paralog LoaP are widely found in Bacillota, Actinomycetota, and Spirochaetota, where they are located near biosynthetic operons for secondary metabolites. Previously, Bacillus velezensis LoaP was shown to promote readthrough of transcription termination sites located within operons encoding for the biosynthesis of the antibiotics difficidin and macrolactin. Thus, some secondary metabolites are produced from terminator-laden operons that rely on antitermination factors such as LoaP. In this study, we show that LoaP bypasses termination by specifically antagonizing the transcription factor NusA. This demonstrates that the LoaP antitermination complex is simpler than expected and that additional factors are not required. Together, our data expand the repertoire of NusG specialized paralogs and the antitermination mechanisms they utilize. Understanding this molecular mechanism will aid in the discovery of natural products regulated by LoaP proteins and assist in the development of new synthetic biology tools that improve the heterologous expression of natural products.

A combination of exoribonucleases and endoribonucleases degrades RNA polymers to recycle nucleoside monophosphates. A byproduct of these reactions is the accumulation of short RNAs, 2–5 nucleotides in length. Characteristic enzymes, generally referred to as nanoRNases, specifically process short RNAs. Genes encoding nanoRNases are essential in some bacteria; therefore, it is assumed that the accumulation of short RNAs is detrimental to cells. However, the substrate preferences and enzymatic mechanisms of the known categories of nanoRNase enzymes have not been equally investigated. The NrnB category of nanoRNases has been particularly understudied. In this study, we identified bacterial NrnB homologs and discovered they can be grouped into three classes of proteins, which can be identified by their characteristic sequence features. Purified representatives of these classes of proteins revealed that they all process RNA substrates from the 3′-terminus. The presence of sequence features at the C-terminus was shown to be diagnostic for general exoribonuclease activity against long RNA substrates, whereas the absence of these C-terminal elements was correlated with proteins that preferentially acted against shorter RNA substrates. Together, these data define members of the overall NrnB family of nanoRNase proteins and identify some of their key features.

Dinucleases of the DEDD superfamily, such as oligoribonuclease, Rexo2 and nanoRNase C, catalyze the essential final step of RNA degradation, the conversion of di- to mononucleotides. The active sites of these enzymes are optimized for substrates that are two nucleotides long, and do not discriminate between RNA and DNA. Here, we identified a novel DEDD subfamily, members of which function as dedicated deoxydinucleases (diDNases) that specifically hydrolyze single-stranded DNA dinucleotides in a sequence-independent manner. Crystal structures of enzyme-substrate complexes reveal that specificity for DNA stems from a combination of conserved structural elements that exclude diribonucleotides as substrates. Consistently, diDNases fail to complement the loss of enzymes that act on diribonucleotides, indicating that these two groups of enzymes support distinct cellular functions. The genes encoding diDNases are found predominantly in genomic islands of Actinomycetes and Clostridia, which, together with their association with phage-defense systems, suggest potential roles in bacterial immunity.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2–5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnB_Bs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5′ terminus of RNA substrates, NrnB acts at the 3′ terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3′-5′ exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

Microcompartments are large, organelle-like structures that help bacteria catabolize targeted metabolites while also protecting the cytosol against highly reactive metabolic intermediates. Their protein shell self-assembles into a polyhedral structure of approximately 100 to 200 nm in diameter.

Bacterial RNases process RNAs until only short oligomers (2–5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2–4 nucleotides in length, all of which were processed from their 5′ terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

Diribonucleotides arise from two sources: turnover of RNA transcripts (rRNA, tRNA, mRNA, and others) and linearization of cyclic-di-nucleotide signaling molecules. In both cases, there appears to be a requirement for a dedicated set of enzymes that will cleave these diribonucleotides into mononucleotides. The first enzyme discovered to mediate this activity is oligoribonuclease (Orn) from Escherichia coli. In addition to being the enzyme that cleaves dinucleotides and potentially other short oligoribonucleotides, Orn is also the only known exoribonuclease enzyme that is essential for E. coli, suggesting that removal of the shortest RNAs is an essential cellular function. Organisms naturally lacking the orn gene encode other nanoRNases (nrn) that can complement the conditional E. coli orn mutant. This review covers the history and recent advances in our understanding of these enzymes and their substrates. In particular, we focus on (i) the sources of diribonucleotides; (ii) the discovery of exoribonucleases; (iii) the structural features of Orn, NrnA/NrnB, and NrnC; (iv) the enzymatic activity of these enzymes against diribonucleotides versus other substrates; (v) the known physiological consequences of accumulation of linear dinucleotides; and (vi) outstanding biological questions for diribonucleotides and diribonucleases.

The shell proteins that comprise bacterial microcompartments (BMCs) can self-assemble into an array of superstructures such as nanotubes, flat sheets, and icosahedrons. Physical characterization of BMCs and these superstructures typically relies on electron microscopy, which decouples samples from their solution context. We hypothesize that an investigation of fluorescently tagged BMCs and shell protein superstructures in vitro with high resolution confocal microscopy will lead to new insights into the solution behavior of these entities. We find that confocal imaging is able to capture nanotubes and sheets previously reported by TEM. Using a combination of fluorescent tags, we present qualitative evidence that these structures intermix with one another in a hetero- and homotypic fashion. Complete BMCs are also able to accomplish intermixing as evidenced by colocalization data. Finally, a simple colocalization experiment suggests that fluorescently modified encapsulation peptides (EPs) may prefer certain shell protein binding partners. Together, these data demonstrate that high resolution confocal microscopy is a powerful tool for investigating microcompartment-related structures in vitro , particularly for colocalization analyses. These results also support the notion that BMCs may intermix protein components, presumably from the outer shell. Importance: Microcompartments are large, organelle-like structures that help bacteria catabolize targeted metabolites, while also protecting the cytosol against highly reactive metabolic intermediates. Their protein shell self-assembles into a polyhedral structure of approximately 100-200 nm in diameter. Inside the shell are thousands of copies of cargo enzymes, which are responsible for a specific metabolic pathway. While different approaches have revealed high-resolution structures of individual microcompartment proteins, it is less clear how these factors self-assemble to form the full native structure. In this study, we show that laser scanning confocal microscopy can be used to study microcompartment proteins. We find that this approach allows researchers to investigate the interactions and potential exchange of shell protein subunits in solution. From this, we conclude that confocal microscopy offers advantages for studying the in vitro structures of other microcompartments, as well as carboxysomes and other bacterial organelles.