Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD.

Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor proteins distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

INTRODUCTION

Transcription elongation is punctuated by pauses that serve important functions in permitting correct folding of structural RNA, efficient coupling of transcription and translation, and ensuring efficient transcription termination at the correct site (Saba et al., 2019). Whilst most pausing events serve an important function, on occasion RNA polymerase (RNAP) is unable to restart transcription and must be removed from the DNA to prevent damaging collisions with the DNA replication machinery or other transcription complexes (Adelman & Lis, 2012; Gupta et al., 2013; Pomerantz & O'Donnell, 2008, 2010, Rocha, 2004). Several systems used to resolve stalled transcription complexes have been characterized; for example, Mfd has been shown to bind to stalled transcription complexes (either a stochastic pause during transcription of structured RNA or at a site of DNA damage), physically removing it from the DNA or restarting it via a RecG‐like ATPase motor domain (Ghodke et al., 2020; Ho et al., 2018; Kang et al., 2021; Le et al., 2018; Ragheb et al., 2021; Shi et al., 2020; Westblade et al., 2010). In B. subtilis RNaseJ1 clears stalled RNAP using a torpedo mechanism (5′–3′ exonuclease activity followed by RNAP displacement) (Sikova et al., 2020), and in Escherichia coli the helicase protein RapA is important in recycling RNAP (Liu et al., 2015). UvrD/PcrA in concert with Gre factors has been reported to act on RNAP stalled at a DNA lesion, binding to the complex and using the energy of ATP hydrolysis to backtrack away from the lesion to allow repair systems access to the damaged DNA (Epshtein et al., 2014; Hawkins et al., 2019), although it now appears that the role of these helicases is in preventing the formation of, and resolving, R‐loops (RNA‐DNA hybrids) that can have a detrimental effect on DNA replication (Urrutia‐Irazabal et al., 2021).

An additional system identified in Gram‐positive bacteria required for recycling stalled transcription complexes involves the action of the motor protein HelD (Wiedermannova et al., 2014). The designation of HelD (also called helicase IV) was originally made for a protein identified in E. coli as a weakly processive 3′–5′ DNA helicase (Wood & Matson, 1987). To avoid confusion with the separate classes of HelD proteins that are the focus of this work, the E. coli protein will be referred to as helicase IV. Based on conserved sequence motifs Helicase IV is a superfamily 1 (SF1) helicase, related to housekeeping helicase UvrD/PcrA (Figure 1). The B. subtilis gene yvgS was assigned the name helD based on limited protein sequence conservation to helicase IV (Wiedermannova et al., 2014), although the proteins differed with respect to domain organization (Koval et al., 2019; Wiedermannova et al., 2014) (Figure 1). Little functional, and no structural information is available for helicase IV, although a model generated by AlphaFold2 (Jumper et al., 2021) enables tentative comparison of UvrD/PcrA, helicase IV, and B. subtilis HelD (Figure 1). Helicase IV and HelD show similarity with UvrD/PcrA around the well‐defined 1A and 2A helicase domains (blue and orange, respectively, Figure 1a), but not in other structural motifs associated with helicase activity (UvrD/PcrA domains 1B and 2B). Both helicase IV and HelD have N‐terminal domains not present in UvrD/PcrA helicases, and helicase IV has a putative 1B domain which may account for its reported helicase activity, whilst in the equivalent 1B domain position HelD contains an unrelated sequence that folds into a novel clamp‐arm (CA) structure important in transcription recycling (Newing et al., 2020; Wiedermannova et al., 2014). Whilst UvrD/PcrA and helicase IV have helicase activity, HelD shows none suggesting it has evolved from an SF1‐type helicase into a transcription recycling factor that utilises the energy from ATP hydrolysis catalysed by its helicase motifs for its transcription‐related activity.

FIGURE 1

Relationship between UvrD/PcrA and helicase IV/HelD proteins. The left side shows scaled linear representations of the domain organization of superfamily 1 (SF1) helicase UvrD/PcrA (top), Escherichia coli helicase IV (middle), and B. subtilis HelD (bottom). A scale bar (amino acids) is shown at the bottom. The right‐hand side shows structures, aligned via their 1A and 2A domains, with domains colored corresponding to the left panels. Top, UvrD (PDB ID 3LFU); middle, helicase IV (AlphaFold2 model, AF2); bottom, HelD (taken from RNAP‐HelD complex PDB ID 6WVK). 1A, B, 2A, and 2B refer to conserved SF1 helicase domains. NTD, SCA, and CA refer to the AlphaFold2 modeled N‐terminal domain of helicase IV and the secondary channel arm and clamp arm of HelD, respectively

Studies on HelD from low G+C (Bacillus subtilis) and high G+C (Mycobacterium smegmatis) Gram‐positives revealed that there are two distinct classes of the enzyme, confirmed by phylogenetic and structural analyses (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020). Class I HelD was described from B. subtilis, whilst the structurally distinct Class II enzyme was identified in M. smegmatis (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020). Class I and II HelDs have similar motor domains but differ in the structure of their arms and the mechanism by which these arms perform the mechanical activity of removing nucleic acids and recycling RNAP (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020).

The recent structures of HelD from B. subtilis and M. smegmatis bound to core RNAP (α2ββ’ω) (Kouba et al., 2020; Newing et al., 2020) are shown in Figure 2a and b, along with the Class I B. subtilis (Figure 2c) and Class II M. smegmatis (Figure 2d) enzymes. HelD has an unusual mode of action dependent on two arms (CA and SCA, Figure 2c and d) attached to the central UvrD‐like ATPase motor domain (Head and Torso, Figure 2c and d), in which nucleic acids are pushed out of the active site whilst the DNA binding clamp and RNA exit channels are simultaneously opened, leading to the release of the stalled RNAP (Newing et al., 2020). This recycling activity is powered by ATP hydrolysis and the mechanical action of the two arms that flank the motor domain. In the Class I HelD, the long SCA (Figure 2a and c) can physically remove nucleic acids from the active site (dotted circle in Figure 2a), whereas in the Class II HelD the SCA is too short, and instead nucleic acid removal is performed by a CA insert called the PCh‐loop (Figure 2b and d) (Kouba et al., 2020; Newing et al., 2020). Recent reports also suggest that some Class II HelDs (from M. abscessus and Streptomyces venezuelae) can confer rifampicin resistance through removal of rifampicin by the PCh‐loop (Hurst‐Hess et al., 2021; Surette et al., 2021).

FIGURE 2

The two known structural classes of HelD. Panel A shows the structure of the B. subtilis RNAP‐Class I HelD complex (PDB ID 6WVK). Panel B shows the M. smegmatis RNAP‐Class II HelD complex (PDB ID 6YYS; state II). RNAP subunits and HelDs are colored identically in both panels with the transparency of the β’ subunit set at 50% so that HelD structures adjacent to the RNAP active site region (dashed circles) can be more easily visualized. Panels C and D show HelD structures in the same orientation as in Panels A and B, with the ATP binding site colored in blue and the PCh‐loop from M. smegmatis HelD colored in yellow (see text for details)

In this work, we take advantage of the recent structural information to compile a detailed phylogenetic analysis of HelD showing that many organisms contain more than one (up to 5) different versions of HelD, that the genes encoding these enzymes are all expressed, that HelD is likely to have been acquired by horizontal gene transfer in Gram‐negative Bacteroides and Gram‐positive Coriobacteria and Acidimicrobiia, and that there is a third Class of HelD found in the Gram‐negative Deltaproteobacteria.

EXPERIMENTAL PROCEDURES

Sequence retrieval and analysis

The sequence of B. subtilis 168 HelD (UniProtKB ID: O32215) was used to search for homologues on 11/11/2020 using the NCBI Conserved Domain Architecture Retrieval Tool (Geer et al., 2002), which identified 13,781 sequences, which were trimmed to 11,821 to remove partial sequences (<600 aa). To aid subsequent analyses, particularly for the study of multiple copies of helD genes, the original sequences were used to search complete reference genomes from the KEGG (https://www.kegg.jp) and JGI (https://jgi.doe.gov) databases. HelD and RpoB sequences retrieved from these complete genomes were used for subsequent phylogenetic studies.

Construction of phylogenetic trees

Selected sequences were aligned using MAFFT (Katoh et al., 2002, 2019) with default settings. Sequence alignments were then trimmed using Gblocks (https://ngphylogeny.fr). The best‐fitting model (LG) was determined using ProtTest 3 (Darriba et al., 2011) and phylogenetic trees were constructed using MrBayes 3.2 (Huelsenbeck & Ronquist, 2001; Ronquist et al., 2012), which were run until the standard deviation was below 0.01. Phylogenetic trees were also made on MEGA‐X (Kumar et al., 2018), using the Maximum Likelihood statistical method with 1000 bootstrap replications, and using RAxML (Stamatakis, 2006) using default settings. All trees had the same topology. Trees were visualised using iTol (Letunic & Bork, 2019).

Transcriptome data and analysis

Gene expression data were obtained from datasets deposited in the Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra) and were: B. subtilis 168 (Revilla‐Guarinos et al., 2020); B. cereus F837/76 (Jessberger et al., 2019); Clostridium perfringens 13 (Soncini et al., 2020); Streptomyces coelicolor A3(2) (Jeong et al., 2016); Mycobacterium smegmatis MC2‐155 (Feng et al., 2020); Myxococcus xanthus DK1622 (SRA accession code: PRJNA516475); Bacteroides vulgatus ATCC8482 (SRA accession code: PRJNA473003). Reads were mapped to the respective reference genome sequences, and gene expression levels were calculated in Genious Prime 2020.2.3 (https://www.geneious.com). Transcript per million (TPM) values were used for comparison of helD expression levels cf. rpoB, and pcrA/uvrD (for S. coelicolor A3(2)).

Structure modeling

RNAP RpoB (β) and RpoC (β’) subunits from M. xanthus DK1622 were modeled in SWISS‐MODEL (Waterhouse et al., 2018) using E. coli RNAP, PDB ID: 6ALF (Kang et al., 2017) as a defined template. The M. xanthus HelD structure was modeled using i‐Tasser (Yang et al., 2015) with output model 1 (C‐score −0.48) selected for presentation in this work. Structural images used in this work were prepared in ChimeraX (Pettersen et al., 2021).

RESULTS AND DISCUSSION

Distribution and phylogeny of HelD

Searching for HelD‐like sequences using the conserved domain architecture retrieval tool (CDART; NCBI) portal identified >13,000 hits. Additional searches using NCBI BLASTP suggest that there are substantially more sequences in the database, but many of these are from incomplete genomes and/or metagenomic sequencing projects, making systematic identification and classification of sequences unfeasible, particularly in cases where an organism carries more than one helD gene (see below). Nevertheless, it is clear that HelD is widely distributed in the eubacteria, especially in the Firmicutes and Actinobacteria phyla of the Gram‐positive eubacterial domain. To date, we have not detected HelD‐like sequences in Archaea or Eucarya. Previously, Newing et al. (Newing et al., 2020) showed that HelD sequences fall into two classes, which was confirmed at the structural and functional level in comparing HelD proteins from the Firmicutes and Actinobacteria (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020). Using a wider range of carefully curated sequences from complete genomes identified from the initial CDART search, an unrooted phylogenetic tree was constructed to enable a more detailed understanding of HelD distribution and phylogeny which was compared against the RNAP RpoB (β) subunit (Figure 3; note different tree scales).

FIGURE 3

Unrooted phlyogenetic trees of HelD (A) and RpoB (B) sequences constructed by Bayesean analysis. Tree scale representing amino acid substitutions per site, and bootstrap probability values (red least, to green most, probable) are on the left. Note that the scales are different for HelD and RpoB trees. The HelD class into which sequences fall is indicated in the outer circles as Class I, ‐II and ‐III. Colored arcs indicate the bacterial classes into which the HelD sequences fall; teal, Firmicutes; pale green, Actinobacteria; purple, Clostridia; orange, Bacteroidia; red, Deltaproteobacteria; brown, Coriobacteria; pale yellow, Acidimicrobilia. Individual organisms and HelD sequences are numbered (largest to smallest) and color‐coded starting clockwise from Bacillus subtilis. Organism numbers with one HelD are numbered in black; two, blue; three, red; four, orange; five, green and are listed as follows with gene identifiers and protein length (aa) in brackets: 1 Bacillus subtilis 168 (BSU_33450, 774aa). 2 Bacillus licheniformis ATCC 14580 (bli_00699, 776aa). 3 Bacillus megaterium DSM 319 (BMD_3869, 772aa). 4 Bacillus cereus ATCC10987 (#1 BCE_3516, 768 aa; #2 BCE_2839, 689 aa). 5 Bacillus anthracis AMES (#1 BA_1040, 776 aa; #2 BA_2814, 689 aa). 6 Bacillus cereus AH187 (#1 BCAH187_A1206, 777 aa; #2 BCAH187_A2861, 689 aa). 7 Bacillus cereus ATCC14579 (BC_1041, 777 aa). 8 Bacillus thuringiensis Bt407 (#1 btg_c11000, 778aa; #2 btg_c29280, 691aa). 9 Lactobacillus plantarum WCFS1 (#1 lpl_0432, 769aa; #2 lpl_0910, 768aa). 10 Lactobacillus rhamnosus GG (#1 lrh_01975, 763aa; #2 lrh_02619, 762aa). 11 Leuconostoc lactis WiKim40 (llf_04535, 788aa). 12 Lactobacillus acidophilus NCFM (lac_1676, 687aa). 13 Carnobacterium inhibens subsp. Gilchinskyi WN1359 (caw_09345, 800aa). 14 Enterococcus faecium Aus0004 (#1 EFAU004_01304, 759 aa; #2 EFAU004_00387, 711 aa). 15 Enterococcus faecium DO (#1 HMPREF0351_10989, 759 aa; #2 HMPREF0351_10397, 711 aa). 16 Olsenella uli DSM 7084 (OLS_0501, 731aa). 17 Atopobium parvulum DSM 20469 (Apar_0360, 736aa). 18 Slackia heliotrinireducens DSM 20476: (Shel_05840 (698aa). 19 Eggerthella lenta DSM 2243(Elen_2835, 716aa). 20 Adlercreutzia equolifaciens DSM 19450 (#1 AEQU_1689, 761aa; #2 AEQU_0484, 733aa). 21 Vagococcus teuberi (vte_03205, 717aa). 22 Enterococcus faecalis V583 (EF_0933, 732 aa). 23 Enterococcus faecalis DENG1 (DENG_00988, 732 aa). 24 Enterococcus faecalis OG1RF (OG1RF_10660, 740 aa). 25 Clostridium beijerinckii NCIMB 8052 (#1 cbe_2947, 755aa; #2 cbe_2724, 745aa; #3 cbe_4782, 724aa). 26 Epulopiscium sp. N.t. morphotype B (EPU_RS03295, 735aa). 27 Clostridioides difficile 630 (CD630_04550, 704 aa). 28 Clostridioides difficile RM20291 (CDR20291_0396, 704 aa). 29 Clostridioides difficile CD196 (CD196_0410, 704 aa). 30 Bacteroides vulgatus ATCC 8482 (BVU_3010 (671aa). 31 Bacteroides caccae ATCC 43185 (CGC64_00555, 683aa). 32 Bacteroides cellulosilyticus WH2 (BcelWH2_01491, 693aa). 33 Bacteroides thetaiotaomicron VPI‐5482 (BT_1890, 686aa). 34 Bacteroides ovatus ATCC 8483 (Bovatus_02598 (687aa). 35 Bacteroides xylanisolvens XB1A (BXY_17560, 687aa). 36 Staphylococcus delphini NCTC12225 (sdp_01978, 681aa). 37 Clostridium botulinuim A ATCC3502 (#1 CBO_2904, 763 aa; #3 CBO_3341, 709 aa). 38 Clostridium botulinuim A ATCC19377 (#1 CLB_2867, 763 aa; #3 CLB_3399, 709 aa). 39 Clostridium botulinuim B1 Okra (#1 CLD_1639, 763 aa; #2 CLD_1179, 709 aa). 40 Clostridium perfringens 13 (#1 CPE_1619, 763 aa; #2 CPE_0599, 706 aa). 41 Clostridium perfringens ATCC13124 (#1 CPF_1872, 763 aa; #2 CPF_0580, 706 aa). 42 Clostridium perfringens SM101 (#1 CPR_1591, 763 aa; #2 CPR_0566 706 aa). 43 Myxococcus xanthus DK 1622 (MXAN_5482, 706aa). 44 Sandaracinus amylolyticus DSM 53668 (#1 DB32_004372, 872aa; #2 DB32_003397, 691aa). 45 Minicystis rosea DSM 2400 (#1 A7982_09686, 743aa; #2 A7982_06548, 703aa). 46 Haliangium ochraceum DSM 14365 (Hoch_0025, 852aa). 47 Sorangium cellulosum So157‐2 (SCE1572_03860, 747aa). 48 Acidobacterium ferrooxidans (Afer_1829, 706aa). 49 Cutibacterium acnes KPA171202 (PPA0733, 753aa). 50 Streptomyces venezuelae (#1 SVEN_2719, 779aa; #2 SVEN_5092, 747aa; #3 SVEN_6029, 722aa; #4 SVEN_4127, 675aa; #5 SVEN_3939; 665aa). 51 Streptomyces coelicolor A3(2) (#1 SCO5439, 755 aa; #2 SCO2952, 744 aa; #3 SCO4316, 681 aa; #4 SCO4195, 680 aa). 52 Ilumatobacter coccineus (#1 aym_09360, 715aa; #2 aym_20540, 654aa). 53 Frankia casuarinae Ccl3 (#1 fra_0952, 829aa; #2 fra_2397, 727aa). 54 Frankia alni ACN14a (#1 fal_1589, 939aa; #2 fal_4723, 877aa; #3 fal_3805; 866aa; #4 fal_4811, 751aa). 55 Nonomuraea sp. ATCC55076 (#1 NOA_23645, 772 aa; #2 NOA_16240, 762 aa; #3 NOA_42280, 715 aa; #4 NOA_08745, 660 aa; #5 NOA_48960, 655 aa). 56 Nocardia brasiliensis O31_020410 (#1 nbr_012985, 776aa; #2 nbr_020410, 731aa; #3 nbr: O3I_005870, 699aa). 57 Kineococcus radiotolerans SRS30216 (#1 kra_3607, 759aa; #2 kra_0164, 684aa). 58 Microbacterium sp. PAMC 28756 (mip_00070, 717aa). 59 Mirobacterium hominis SJTG1 (mhos_01135, 744aa). 60 Nocardia farcinica IFM10152 (#1 NFA_19060, 765aa; #2 NFA_44160, 726aa). 61 Mycobacterium smegmatis MC2 155 (MSMEG_2174, 736aa). 62 Rhodococcus sp. 008 (#1 rhod_26990, 760aa; #2 rhod_09075, 731aa). 63 Mycobacterium sp. JS623 (Mycsm_03949, 732aa). 64 Mycolicibacterium phlei (MPHL_03003, 726aa). 65 Mycobacteroides abscessus ATCC 19977 (MAB_3189c, 753aa). 66 Rhodococcus equi 103S (#1 REQ_25070, 759aa; #2 REQ_15310, 739aa). 67 Nocardia asteroides NCTC11293 (#1 nad_03000, 753; #2 nad_04408, 735aa). 68 Leifsonia xyxli subsp. Xyli CTCB07 (Lxx_20770, 787aa). 69 Bifidobacterium longum NCC2705 (BLO_1314, 759aa). 70 Bifodobacterium bifidum PRL2010 (bbp_0546, 759aa). 71 Brevibacterium linens BS258 (bly_10570, 743aa). 72 Brevibacterium flavum ZL‐1 (bfv_07580, 755aa). 73 Corynebacterium glutamicum ATCC13031 (CG_1555, 755aa). 74 Corynebacterium diptheriae NTCC13129 (DIP_1156, 770aa). 75 Rhodococcus rhodochrous NCTC10210 (rrt_02795, 772aa). Nonomuraea sp. ATCC55076 (55), Nocardia brasiliensis O31_020410 (56) and Nocardia farcinica IFM10152 (60) contain two copies of the rpoB gene (numbered x.a and x.b in panel B). Copy 1 is the housekeeping rpoB and copy 2 is a rifamipicin‐resistant rpoB expressed during antibiotic production in those organisms.

Four features are clear from this tree (Figure 3a): 1. HelD is also present in Gram‐negative bacteria; 2. The third class of HelD is present in the Deltaproteobacteria; 3. In some organisms HelD has been ancestrally acquired by horizontal gene transfer; 4. Many organisms contain more than one helD gene, with the Firmicutes, Clostridia, Acidimicrobiia, and Deltaproteobacteria having up to three, and the Actinobacteria up to five.

Overall, the tree contains three major branches: Class I HelD sequences originating mainly from the low G+C Gram‐positives and Bacteriodia, Class II HelD sequences from the high G+C Gram‐positives, and a novel Class III identified in Deltaproteobacteria. Interestingly, the HelD sequences from the Actinobacterial Coriobacteria class, typified by Olsenella uli that is associated with gingivitis, are all located to the Class I branch of the tree (numbers 16–20; Figure 3). Branch divergence and clustering of sequences to regions of the tree comprising Lactobacilli (numbers 14, 15, 21–24; Figure 3) and Clostridia (numbers 25–29; Figure 3) indicate that an ancestral Coriobacteria likely acquired helD genes by horizontal gene transfer from these organisms (Appendix 1; Figure A1). That Coriobacteria is isolated from the gingival crevice, gastrointestinal and genital tracts (Clavel et al., 2014) is consistent with this proposition. The length of the branches suggests this horizontal transfer event occurred long ago but after the evolution of the mammalian hosts that provide environments with co‐localised Lactobacilli, and that helD genes have been stably inherited and co‐evolved within the Coriobacteria. In addition to the helD gene from Adlercreutzia equolifaciens DSM 19450 (AEQU_1689, number 20.1; Figure 3) that clusters with those of the other Coriobacteria, A. equolifaciens contains a second helD gene (AEQU_0484, number 20.2; Figure 3) that clusters with Clostridia, suggesting it may have been acquired through a separate horizontal gene transfer event rather than through duplication and evolution of a gene inherited by a single acquisition event (Appendix 1; Figure A1). The fact that Lactobacilli, Clostridia, and Aldercreutzia all inhabit the gastrointestinal tract makes this a reasonable hypothesis. There is also some evidence that Class II HelD sequences have been acquired by horizontal gene transfer between the Actinobacteria to the Acidimicrobiia (numbers 48, 52.1, and 52.2; Figure 3 and Appendix 1 Figure A2). The Acidimicrobiia is a recently described class, exemplified by Acidobacterium ferrooxidans (number 48; Figure 3) that have been isolated from diverse, but generally acidic and hostile environments, and tend to grow slowly which may account for the paucity of information and diversity of species currently available. At least one species of the Acidimicrobiia, Ilumatobacter coccineus (number 52, Figure 3) contains multiple copies of helD.

FIGURE A1

Acquisition of helD genes by Coriobacteria from Firmicutes and Clostridia. The phylogenetic tree from Figure 1 is shown on the left side with the region boxed expanded on the right side. Bacterial classes are colored, species numbered, number of helD genes colored as in Figure 1: Bacilli, teal; Clostridia, purple; Coriobacteria, brown. 14 Enterococcus faecium Aus0004 (#2 EFAU004_00387, 711 aa). 15 Enterococcus faecium DO (#2 HMPREF0351_10397, 711 aa). 16 Olsenella uli DSM 7084 (OLS_0501, 731aa). 17 Atopobium parvulum DSM 20469 (Apar_0360, 736aa). 18 Slackia heliotrinireducens DSM 20476: (Shel_05840 (698aa). 19 Eggerthella lenta DSM 2243(Elen_2835, 716aa). 20 Adlercreutzia equolifaciens DSM 19450 (#1 AEQU_1689, 761aa; #2 AEQU_0484, 733aa). 21 Vagococcus teuberi (vte_03205, 717aa). 22 Enterococcus faecalis V583 (EF_0933, 732 aa). 23 Enterococcus faecalis DENG1 (DENG_00988, 732 aa). 24 Enterococcus faecalis OG1RF (OG1RF_10660, 740 aa). 25 Clostridium beijerinckii NCIMB 8052 (#2 cbe_2724, 745aa). 26 Epulopiscium sp. N.t. morphotype B (EPU_RS03295, 735aa). 27 Clostridioides difficile 630 (CD630_04550, 704 aa). 28 Clostridioides difficile RM20291 (CDR20291_0396, 704 aa). 29 Clostridioides difficile CD196 (CD196_0410, 704 aa). One helD gene, black; two, blue; three, red

FIGURE A2

Acquisition of helD genes by Acidimicrobiia from Actinobacteria. The phylogenetic tree from Figure 1 is shown on the left side with the region boxed expanded on the right side. Bacterial classes are colored, species numbered, number of helD genes colored as in Figure 1: Actinobacteria, pale green; Acidimicrobiia, pale yellow. 48 Acidobacterium ferrooxidans (Afer_1829, 706aa). 49 Cutibacterium acnes KPA171202 (PPA0733, 753aa). 50 Streptomyces venezuelae (#1 SVEN_2719, 779aa; #2 SVEN_5092, 747aa; #3 SVEN_6029, 722aa; #4 SVEN_4127, 675aa; #5 SVEN_3939; 665aa). 51 Streptomyces coelicolor A3(2) (#1 SCO5439, 755 aa; #2 SCO2952, 744 aa; #3 SCO4316, 681 aa; #4 SCO4195, 680 aa). 52 Ilumatobacter coccineus (#1 aym_09360, 715aa; #2 aym_20540, 654aa). 53 Frankia casuarinae Ccl3 (#1 fra_0952, 829aa; #2 fra_2397, 727aa). 54 Frankia alni ACN14a (#1 fal_1589, 939aa; #2 fal_4723, 877aa; #3 fal_3805; 866aa; #4 fal_4811, 751aa). 55 Nonomuraea sp. ATCC55076 (#1 NOA_23645, 772 aa; #2 NOA_16240, 762 aa; #3 NOA_42280, 715 aa; #4 NOA_08745, 660 aa; #5 NOA_48960, 655 aa). 56 Nocardia brasiliensis O31_020410 (#1 nbr_012985, 776aa; #2 nbr_020410, 731aa; #3 nbr: O3I_005870, 699aa). 57 Kineococcus radiotolerans SRS30216 (#1 kra_3607, 759aa; #2 kra_0164, 684aa). 58 Microbacterium sp. PAMC 28756 (mip_00070, 717aa). 59 Mirobacterium hominis SJTG1 (mhos_01135, 744aa). 60 Nocardia farcinica IFM10152 (#1 NFA_19060, 765aa; #2 NFA_44160, 726aa). 61 Mycobacterium smegmatis MC2 155 (MSMEG_2174, 736aa). 62 Rhodococcus sp. 008 (#1 rhod_26990, 760aa; #2 rhod_09075, 731aa). 63 Mycobacterium sp. JS623 (Mycsm_03949, 732aa). 64 Mycolicibacterium phlei (MPHL_03003, 726aa). 65 Mycobacteroides abscessus ATCC 19977 (MAB_3189c, 753aa). 66 Rhodococcus equi 103S (#1 REQ_25070, 759aa; #2 REQ_15310, 739aa). 67 Nocardia asteroides NCTC11293 (#1 nad_03000, 753; #2 nad_04408, 735aa). 68 Leifsonia xyxli subsp. Xyli CTCB07 (Lxx_20770, 787aa). 69 Bifidobacterium longum NCC2705 (BLO_1314, 759aa). 70 Bifodobacterium bifidum PRL2010 (bbp_0546, 759aa). 71 Brevibacterium linens BS258 (bly_10570, 743aa). 72 Brevibacterium flavum ZL‐1 (bfv_07580, 755aa). 73 Corynebacterium glutamicum ATCC13031 (CG_1555, 755aa). 74 Corynebacterium diptheriae NTCC13129 (DIP_1156, 770aa). 75 Rhodococcus rhodochrous NCTC10210 (rrt_02795, 772aa). One helD gene, black; two, blue; three, red; four, orange; five, green

Comparison of the phylogenetic tree of the RNA polymerase β subunit RpoB with the HelD tree supports this assumption that helD genes in the Coriobacteria and Acidimicrobiia have been acquired by horizontal gene transfer from Firmicutes/Clostridia/Actinobacteria that share the same ecological niches (Figure 3a and b). Acquisition of helD genes by horizontal gene transfer in the Bacteroidia is described below.

Acquisition of helD in Gram‐negative Bacteroides

HelD sequences were also identified in the phylum of Gram‐negative bacteria, Bacteroides. Mapping sequences to the phylum Bacteroidota shows HelD is widely distributed throughout the class Bacteroidia with additional representation in the classes Rhodothermia and Ignavibacteria (Appendix 1; Figure A3). Phylogenetically, many of the Bacteroidial HelD sequences clustered close to HelD sequences from Clostridioides difficile (Figure 3a and Appendix 1 Figure A4; sequences 27–29 C. difficile, 30–35 Bacteroides). Extended analysis indicated that HelD sequences from Bacteroides and Parabacteroides (family Porphyromonadaceae) clustered closest to those from Firmicutes that are strict gut anaerobes from the order Clostridiales (Appendix 1; Figure A5). These bacteria were from cluster IV (Ruminoccoaceae) and XIVa (Lachnospiraceae) that are abundant gut microbes associated with many aspects of good health, and the cluster XI gut pathogen C. difficile (Lopetuso et al., 2013; Lozupone et al., 2012; Milani et al., 2017). Since the Bacteroides and Parabacteroides are also abundant obligate gut anaerobes, this clustering suggested that helD was horizontally transferred from an anaerobic gut Firmicute, most likely from the order Clostridiales (Appendix 1; Figure A5). Analysis of the genome context of helD genes indicated they were not (or are no longer) located in mobile genetic elements, except for B. thetaiaotamicron, and along with their widespread distribution in Bacteroides/Parabacteroides suggests helD genes have been retained over a significant period, indicating they serve a useful cellular function. The fact that HelD sequences identified in Bacteroides cluster with Class I sequences from the low G+C Gram‐positive bacteria rather than forming a separate Class, as seen with HelD from the Deltaproteobacteria (see below), further supports the idea that this group acquired helD genes by horizontal gene transfer due to sharing a similar environmental niche to anaerobic gut Clostridiales.

FIGURE A3

Distribution of helD genes in the phylum Bacteroidota. HelD sequences from Bacteroidota RefSeq genomes were retrieved from a BLASTP search and mapped to individual species within the phylum Bacteroidota using Annotree (Mendler et al.,2019). Bacteroidotal classes are shown in the colored outer ring with Bacteroidia in pink, Rhodothermia in grey, Chlorobia in light grey, UBA10030 in lime green, Kryptonia in pale green, Ignavibacteria in cyan, Kapabacteria in pale blue, and SZUA‐365 in blue. Individual species are shown as lines radiating out from the circular dendrogram with species containing HelD sequences highlighted in bright blue

FIGURE A4

Acquisition of helD genes by Bacteroides from Clostridia. The phylogenetic tree from Figure 1 is shown on the left side with the region boxed expanded on the right side. Bacterial classes are colored, species numbered, number of helD genes colored as in Figure 1: Bacilli, teal; Clostridia, purple; Bacteroides, orange; Coriobacteria, brown. 20 Adlercreutzia equolifaciens DSM 19450 (#2 AEQU_0484, 733aa). 25 Clostridium beijerinckii NCIMB 8052 (#2 cbe_2724, 745aa; #3 cbe_4782, 724aa). 26 Epulopiscium sp. N.t. morphotype B (EPU_RS03295, 735aa). 27 Clostridioides difficile 630 (CD630_04550, 704 aa). 28 Clostridioides difficile RM20291 (CDR20291_0396, 704 aa). 29 Clostridioides difficile CD196 (CD196_0410, 704 aa). 30 Bacteroides vulgatus ATCC 8482 (BVU_3010 (671aa). 31 Bacteroides caccae ATCC 43185 (CGC64_00555, 683aa). 32 Bacteroides cellulosilyticus WH2 (BcelWH2_01491, 693aa). 33 Bacteroides thetaiotaomicron VPI‐5482 (BT_1890, 686aa). 34 Bacteroides ovatus ATCC 8483 (Bovatus_02598 (687aa). 35 Bacteroides xylanisolvens XB1A (BXY_17560, 687aa). 36 Staphylococcus delphini NCTC12225 (sdp_01978, 681aa). 37 Clostridium botulinuim A ATCC3502 (#3 CBO_3341, 709 aa). 38 Clostridium botulinuim A ATCC19377 (#3 CLB_3399, 709 aa). 39 Clostridium botulinuim B1 Okra (#2 CLD_1179, 709 aa). 40 Clostridium perfringens 13 (#2 CPE_0599, 706 aa). 41 Clostridium perfringens ATCC13124 (#2 CPF_0580, 706 aa). 42 Clostridium perfringens SM101 (#2 CPR_0566 706 aa). One helD gene, black; two, blue; three, red

FIGURE A5

Phylogenetic tree of HelD sequences from Bacteroides and Clostridia. Tree scale and bootstrap values are shown at the top and left, respectively. Colored boxes denote cluster IV and XIVa Clostridia (yellow), cluster IX Clostridia (purple), and Bacteroides (red). The numbers in parentheses correspond to the organisms used in Figure 1. Roseburia intestinalis (Rosebur intest), Blautia sp. SG‐772 (Blaut SG772), Blautia sp. N6H1‐15 (Blaut N6H1‐15b), Pseudoflavonifractor sp. BSD2780061688st1 E11 (PseudoflavonB), Ruminococcus lactaris (Rumino lact), Anaerotruncus sp. 1XD22‐93 (Anaerotruncus 1XD22‐93), Ruminococcus gnavus (Rumino gnavus), Coprococcus comes (Copro comes), B. vulgatus ATCC 8482 (BVU 3010), B. caccae ATCC 43185 (CGC64 00555), B. cellulosilyticus WH2 (BcelWH2 01491), B. ovatus ATCC 8483 (Bovatus 02598), B. xylanisolvens XB1A (BXY 17560), C. difficile 630 (CDif 630), C. difficile RM20291 (CDif R20291), C. difficile CD196 (CDif CD196), Faecalicatena contorta (Faecal cont), Caproiciproducens sp. NJN‐50 (Caproiciproducens NJN‐50), Eubacterium uniforme (Eubact uncl), Hungatella hathewayi (Hungatel hath), Eubacterium limosum (Eubact limo), Faecalicatena orotica (Faecal orot), Blautia marasmi (Blaut maras), Enterocloster bolteae (Enterocl boltA and B), Clostridium symbiosum (Clost symbio), and Enterocloster citroniae (Enterocl citro)

A novel HelD class in Gram‐negative bacteria

The analysis presented in this work also shows that there is a third class of HelD proteins encoded by the Deltaproteobacteria (Class III, Figures 3 and 4; see below). Newing et al. (Newing et al., 2020) identified Class I and II HelD proteins based on the conservation of twelve sequence motifs. These motifs (labeled I‐XII, Appendix 1; Figure A6) are all conserved in Class III proteins (exemplified by Myxococcus xanthus HelD), despite the low overall levels of sequence similarity found in HelD proteins (Newing et al., 2020). A model of M. xanthus HelD was also generated from an unbiased screen of the protein structure database (Figure 4; see Materials and Methods). As seen with Class I and II proteins, there is a HelD‐specific N‐terminal domain of ~50–150 amino acids that has a long antiparallel α‐helical structure (secondary channel arm, SCA, Figure 4b) that is required to anchor HelD in the secondary channel of its cognate RNAP (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020), and the 1A helicase domain is split by the insertion of an arm‐like structure (clamp arm, CA, Figure 4b and S6) that is used to bind within the primary channel of RNAP, forcing it open to aid the release of bound nucleic acids (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020).

FIGURE 4

Three classes of HelD. Panel A shows a focused unrooted phylogenetic tree constructed using HelD sequences, with numbers (#) as used in Figure 1A: B. subtilis 168, BSU [#1]; C. perfringens 13, CPE [#40]; S. coelicolor A3(2), SCO [#51]; M. smegmatis MC2 155, MSMEG [#61], and Deltaproteobacterial sequences from M. xanthus DK 1622, MXAN [#43]; S. amylolyticus DSM 53668, DB32 [#44]; M. rosea DSM 2400, A7982 [#45]; H. ochraceum DSM 14365, Hoch [#46]; S. cellulosum So157‐2, SCE1572 [#47]. Tree scale representing amino acid substitutions per site, and bootstrap values are shown on the left. The coloring of bacterial classes is the same as in Figure 1. Panel B shows structures (ribbons and transparent surface representations) of whole HelD (top) and Trp‐cage regions (bottom) of Class I (B. subtilis PDB ID 6WVK), Class II (M. smegmatis PDB ID 6YYS), and Class III (M. xanthus, homology model) using the same color scheme for bacterial classes as in Figures 1 and 2A. Conserved Trp (all classes) and additional amino acid (Class III) are shown as green sticks, with other amino acids that form the cage shown in the appropriate color for their class

FIGURE A6

Conserved HelD sequence motifs. Panel A shows a schematic of B. subtilis HelD domain organization with conserved sequence motifs adapted from Newing et al., (Newing et al., 2020), with panel B showing the equivalent sequence motifs from M. xanthus HelD. Appendix Table A1 shows the conserved sequence motifs with sequence numbers referring to the B. subtilis HelD sequence. X corresponds to a poorly conserved sequence (any amino acid) and h to a conserved hydrophobic residue. Residues colored red are specific to class I and green to class II sequences. The HelD motifs from the Class III M. xanthus HelD (Class IIIMX) are shown in the right column with absolutely conserved motif residues shown in purple (blue for the ATP binding motifs) and the Class III defining residue (F in the case of M. xanthus) that is inserted in the DWRAP motif shown in grey (see text for more details)

An absolutely conserved DWR (Asp‐Trp‐Arg) sequence motif was identified in the unique N‐terminal domain of all HelD sequences, and determination of the structures of HelD showed that the conserved Trp residue resides within a hydrophobic pocket called the Trp‐cage, important in stabilizing the interaction between the N‐terminal domain wedged deep into the secondary channel of RNAP and the helicase 1A domain (Newing et al., 2020). In most HelD sequences identified to date, the DWR motif is extended to DWR[A/S]P, but in Deltaproteobacterial HelDs there is an additional amino acid inserted in this motif following the R residue, i.e., DWRX[A/S]P, which is a key defining feature of a Class III HelD (Appendix 1; Figure A6). This additional amino acid does not appear to be highly conserved, the motif being DWRFAP in M. xanthus, DWRNAP in Haliangum ocraceum, and DWRHAP in Sorangium cellulosum, with H or N appearing to be most common. Modeling suggests this amino acid is located on a loop with its side chain in an additional pocket that may be important in reinforcing the connection between the SCA and torso, potentially through burying the conserved Trp deeper inside the Trp‐cage in comparison with Class I and II HelDs (boxed green residues, Figure 4b). Structural modeling also shows the SCA of M. xanthus HelD (HelDMX) is longer than that of M. smegmatis (HelDMS) but shorter than the B. subtilis protein (HelDBS). The tip of the SCA of HelDMX does not reach the active site (catalytic Mg2+, green sphere; compare dashed circles in Figure 5c–f) but would clash with the bridge helix in RNAP (teal, Figure 5d and f), potentially causing it to distort and displace the template DNA strand as seen with HelDBS (Newing et al., 2020). The RNAP trigger loop contains a large insertion in the Deltaproteobacteria (β’In6, Figure 5b) similar to that seen in Gammaproteobacteria, and it was assumed this (and the βIn4 insertion, Figure 5b) would sterically interfere with HelD binding to RNAP in Gram‐negative bacteria. Although the trigger loop in the modeled M. xanthus RNAP–HelD complex does clash with HelDMX (Figure 5e and f), this is not extensive, and given the inherent flexibility in this domain, small conformational changes would readily enable binding as seen in Gram‐positive bacteria (Kouba et al., 2020; Newing et al., 2020; Pei et al., 2020). The CA of HelDMX is similar in size to that of HelDMS (although it does not contain a PCh domain; Figure 4b). The CA domain is required for clamp opening and DNA release in the Gram‐positive systems, and likely will serve a similar function in Class III HelDs.

FIGURE 5

Comparison of B. subtilis RNAP–HelD complex with the M. xanthus model. Panels A and B show structures of B. subtilis (PDB ID 6WVK) and M. xanthus (model) RNAPs in complex with HelD, respectively, in which HelD has been removed to more clearly visualize elements referred to in the text. The trigger loop (yellow) and bridge helix (teal) are indicated along with the lineage‐specific βIn4 (also yellow) and β’In6 inserts in the M. xanthus model. Panels C and E show the B. subtilis RNAP–HelD complex. Panels D and F show M. xanthus RNAP–HelD model. RNAP is shown in grey in all panels, HelD in red, bridge helix in teal, and trigger loop in yellow (see text for further details). The active site Mg2+ is shown as a small green sphere (within the dotted circles). The arrows in panels C and E denote the view of the respective RNAP–HelD complex in panels E and F. The view in panels C and D is into the primary channel to which the clamp arm (CA) of HelD binds. The view in panels E and F is into the secondary channel (dotted circle) into which the secondary channel arm (SCA) is inserted

Examination of sequences retrieved from the CDART search indicated helD genes may be even more widely distributed in the Proteobacteria (including the Gammaproteobacteria), although this could not be verified by searches of complete genomes in databases such as KEGG and may represent misclassification from metagenomic sequencing projects. For example, BLASTP searches suggest hits reported as being from E. coli and Vibrio vulnificus identified from metagenomic data are in fact from Bacteroides and Bacillus, respectively (Poyet et al., 2019), and NCBI SRA accession code: PRJNA523266). Nevertheless, helD genes may be more widely distributed in Proteobacteria.

RNAP δ subunit and HelD

The Firmicutes have the smallest multi‐subunit RNAPs currently known (Lane & Darst, 2010a, 2010b), as well as auxiliary subunits δ and ε that are not found in other bacteria (Keller et al., 2014; Weiss & Shaw, 2015). In the original work characterizing the function of HelD as a transcription complex recycling factor, it was shown that although δ or HelD on their own enhanced recycling, there was a synergistic relationship between them in B. subtilis transcription recycling assays (Wiedermannova et al., 2014). Structural analysis of RNAP recycling complexes shows that δ and HelD interact, as well as providing clues as to how δ could enhance the recycling activity of HelD by augmenting clamp opening (Pei et al., 2020). These structural studies also provided insights into how δ could facilitate transcription recycling in the absence of HelD (Miller et al., 2021). Genome searches indicated that not all Firmicutes contained both helD and rpoE (encoding the δ subunit) genes, and an analysis was performed based on the rpoB gene to establish whether there is segregation of genes amongst orders and/or based on the natural environment (Figure 6).

FIGURE 6

Phylogenetic tree of RpoB with respect to the distribution of HelD and the d subunit of RNAP. Tree scale and bootstrap values are shown on the left. Organisms that contain the d subunit (DELTA) are shown in red, just HelD (blue) and both d and HelD (black). Mycoplasma mycoides (MycoMyco), Mycoplasma capricolum (MycoCapr), Mycoplasma pneumoniae (MycoPneu), Mycoplasma marinum (MycoMari), Erysipelatoclostridium cocleatum (ClosCocl), Erysipelatoclostridium inoccuum (ClosInno), Clostridioides difficile (ClosDiff), Clostridium botulinum (ClosBotu), Clostridium perfringens (ClosPerf), Clostridium sartagoforme (ClosSart), Clostridium beijernickii (ClosBeij), Bacillus subtilis (BacSu), Staphylococcus lentus (StapLent), Staphylococcus equorum (StaphEquo) Staphylococcus saprophyticus (StapSapr), Staphylococcus aureus (StapAure), Staphylococcus felis (StapFeli), Staphylococcus agnetis (StapAgne), Staphylococcus rostri (Staprost), Staphylococcus pseudointermidius (StapPseu), Staphylococcus delphini (StapDelp), Enterococcus gallinarum (EnteGall), Enterococcus cecorum (EnteCeco), Enterococcus plantarum (EntePlan), Enterococcus mundti (EnteMundt), Enterococcus faecalis (EnteFaec), Enterococcus faecium (EnteFium), Streptococcus pneumoniae (StrepPneu), Streptococcus marmotae (StrepMarmo), Streptococcus suis (StrepSuis), Streptococcus thermophilus (StrepTherm), Streptococcus agalactiae (StrepAgala), Streptococcus pyogenes (StrepPyog), Streptococcus canis (StrepCani), Lactobacillus sakei (LactSake), Lactococcus rodentium (LactRode), Lactobacillus johnsonii (LactJohn), Lactobacillus salivarius (LactSali), Lactobacillus plantarum (LactPlan), Lactobacillus fermentum (LactFerm), Lactobacillus brevis (LactBrev), Leuconostoc pseudomesenteroides (LeucPseu), Leuconostoc mesenteroides (LeucMese), Leuconostoc sp. (Leuconos), and Leuconostoc lactis (LeucLact)

In the bulk of cases, the Bacilli, Lactobacilli, Leuconostoc, and Enterococci contained genes for both HelD and δ, and if the gene for one protein was missing, the other was present (Figure 6). The Staphylococci were heterogeneous with species such as S. rostri containing both helD and rpoE genes, whereas S. aureus only contained the gene for the δ subunit. There is a segregation of species containing both helD and rpoE cf. rpoE only, with rpoE only present in the S. saprophyticus and S. aureus clusters (Takahashi et al., 1999). Species that fall within the S. hyicus‐intermedius cluster (e.g., S. rostri) contained both helD and rpoE, but there were exceptions such as S. felis, which only contained rpoE (Figure 6). The Streptococci (order Lactobacillales) only contained the rpoE gene (Figure 6), whereas the Clostridia, except for C. (Erysipelatoclostridium) cocleatum and inoccuum, only contained helD genes (Figure 6). Thus, it appears that in the Firmicutes, especially class Bacillus, the default situation is for both rpoE and helD to be present, but the absence of one gene is compensated for by the presence of the other to ensure the ability to recycle stalled transcription complexes is retained.

Many bacteria contain multiple helD genes

A striking observation made in the preliminary phylogenetic analysis of HelD was that some organisms contain more than one helD gene (Newing et al., 2020). This preliminary analysis has now been extended and it is clear that the presence of >1 helD is common and is found in both Gram‐positive and ‐negative organisms (Figure 3a). Using complete genome sequences, up to 5 genes encoding HelD have been identified (e.g. Nonomuraea sp. ATCC55076 [organism 55]; Figure 3a and Appendix 1; Figure A7), and organisms have been identified with 1, 2, 3, 4, or 5 helD genes. Although most contain a single helD gene, low G+C Gram‐positives and Gram‐negatives were not found with >3, and high G+C Gram‐positive Actinobacteria such as Streptomyces, Nonomuraea, and Frankia were identified with ≥4 helD genes. A simple assumption is that these multiple genes are the product of amplification through recombination, and this may well be the root of their original source, but phylogenetic analysis indicates each gene is unique, and organisms with more than one helD gene tend to encode both large (~740–850 aa) and small (~680–720 aa) variants. The variation in sequence length is due to differences in the flanking SCA and CA domains (arms) with the core 1A and 2A helicase domains all being of similar size. This suggests the motor function of these proteins is conserved, but the function of large vs small HelD variants may differ depending on the size of the SCA and CA arms. The multiple helD genes also segregate to Class I, ‐II, or ‐III according to the organism in which they are found; Class I sequences are found in Firmicutes, whereas Actinobacteria all have Class II sequences (except for the Coriobacterium Adlercreutzia equolifaciens, above), and Class III sequences are found in Deltaproteobacteria. Of the Bacteroides/Parabacteroides analyzed to date, all encode only a single Class I helD gene.

FIGURE A7

Distribution of the five helD genes from Nonomuraea sp. ATCC55076. The phylogenetic tree from Figure 1 is shown unannotated apart from boxing the region corresponding to the Actinobacteria pale green, and indicating the location of the Nonomuraea helD genes: #1 NOA_23645, 772 aa; #2 NOA_16240, 762 aa; #3 NOA_42280, 715 aa; #4 NOA_08745, 660 aa; #5 NOA_48960, 655 aa

Some or all of the additional helD sequences might have represented cryptic genes that are not expressed under any conditions, or that they are differentially expressed during different growth phases or conditions, which might provide clues to potential functions. Transcriptomics data were retrieved from the Sequence Read Archive (SRA) for selected organisms containing 1 or >1 helD representative of all three classes of HelD, and expression levels compared relative to rpoB (RNAP β subunit) and another housekeeping gene (SF1 helicase pcrA/uvrD). In all cases, all of the helD genes were expressed, often at an approximately similar level to pcrA/uvrD (Figure 7). The RNA‐seq data of B. subtilis helD and pcrA obtained from experiments by Revilla‐Guarinos et al. (Revilla‐Guarinos et al., 2020) to examine changes in gene expression in a model soil organism on exposure to the antifungal agent amphotericin B produced by Streptomycetes closely matched that of the oligonucleotide hybridization transcriptomics data of Nicolas et al. (Nicolas et al., 2012) and showed the level of helD expression was not influenced by amphotericin B and was ~3% that of rpoB (Figure 7a). This is also consistent with proteomics analysis indicating HelD is present at ~6% the level of RNAP (Delumeau et al., 2011). B. cereus contains two helD genes and the data set from strain F837/76 (Jessberger et al., 2019) grown in the presence and absence of mucin that can influence toxin production shows that both copies (one large, one small variant) are expressed, albeit at low levels, and expression is not significantly affected on exposure to mucin (Figure 7b). C. perfringens also contains two Class I helD genes, labeled CPE_0599 (small; 706 aa) and CPE_1619 (large; 763 aa) in strain 13, and expression levels were determined from datasets of cells grown in brain heart infusion (BHI) and a rich medium developed for the optimal growth of fastidious anaerobes, fastidious anaerobe broth +2% glucose (FABG) medium (Soncini et al., 2020). Both genes were expressed at levels comparable to helD in B. subtilis, and their cognate prcA/uvrD, although CPE_0599 expression increased ~3‐fold and CPE_1619 expression decreased in FABG medium compared to BHI medium (Figure 6c).

FIGURE 7

Expression levels of HelD. The relative transcript levels of helD and pcrA/uvrD compared to rpoB are shown in panels A–G. Organism names are shown on the top of each plot and gene expression levels are color‐coded according to the keys below the plots. The sizes of the HelD isoforms in amino acids are indicated above the corresponding column in each panel. Details of the sources of the data sets used are provided in the text. A. B. subtilis 168 data; control teal, amphotericin B (AmpB) treatment red. B. B. cereus F837/76 data; control teal, mucin treatment red. C. C. perfringens 13 data; growth in brain heart infusion (BHI) teal, fastidious anaerobic broth +glucose (FABG) red. D. S. coelicolor A3(2) data; mid‐exponential growth (M) teal, transition phase (T) red, late exponential (L) blue, stationary phase (S) green. E. M. smegmatis MC2‐155 data; control teal, greA deletion strain (ΔGreA) red. F. M. xanthus DK1622 data; vegetative growth teal, 12 h after initiation of sporulation (T12) red. G. B. vulgatus ATCC 8482 data; control teal, supplemented with thiamine (Thi) red

S. coelicolor A2(3) contains four Class II helD genes, two encoding large (SCO_2952 744 aa, and SCO_5439 755 aa) and two encoding small (SCO_4195 680 aa, and SCO_4316 681 aa) variants. Data from a study on growth phase‐dependent changes in gene expression (Jeong et al., 2016) were obtained from the SRA for analysis of helD expression and compared with rpoB and pcrA. All four helD genes were expressed with relative levels changing ~2‐fold dependent on the growth phase (Figure 7d). Expression levels were generally highest during mid‐log and transition, and lowest during late and stationary phases, with modest changes between the ratios of expression of the different gene copies at all stages. The RNA‐seq data set for M. smegmatis comparing changes in gene expression on the deletion of the transcript cleavage factor GreA that is important in rescuing back‐tracked RNAP (Feng et al., 2020) showed that expression of the single helD gene was substantially higher than in most other organisms, at about 25% the level of rpoB suggesting HelD may be particularly abundant in the Mycobacteria (Figure 7e). The expression levels of helD were similar in the presence and absence of greA indicating each factor acts on stalled transcription complexes independently of each other.

Analysis of RNA‐seq data showed helD genes were also expressed in Gram‐negative M. xanthus and B. vulgatus (Figure 7f and g), showing that despite the structural differences adjacent to the HelD interaction sites in the β and β’ subunits of RNAP from these organisms, HelDs are expressed and likely able to bind and functionally interact with their cognate RNAPs. The data for M. xanthus were obtained to examine changes in gene expression during the development of fruiting bodies and spores. It is interesting to note that expression of helD in M. xanthus increases during the development of spores (not to be confused with sporulation in the Firmicutes) and may point to a role in the storage of inactive RNAP during dormancy as has been proposed for B. subtilis HelD (Pei et al., 2020). The study in B. vulgatus was designed to investigate the effect on gene expression of exogenous thiamine that may be important in niche establishment in the gut. Therefore, in most/all organisms that contain helD gene(s), it/they are expressed. The reason why one organism contains a single gene and closely related species contain more than one (e.g. B. subtilis and B. cereus, Figure 6a and b) is currently not clear, but the expression data would suggest that each isoform has a functional role to play in the cell, and there is not a significant difference in the expression of large vs small helD variants.

CONCLUSIONS

In this work, we have examined the phylogenetic distribution and classification of the transcription recycling factor HelD in detail and have identified a new class restricted to the Deltaproteobacteria. In addition, it appears helD genes have been acquired by horizontal transfer on at least three occasions; Bacteroides have acquired helD from the Clostridiales, whereas the Coriobacteria have acquired it from the Lactobacilli and Clostridiales. The gut microbiome is known as an environment conducive to horizontal gene transfer, especially with respect to the distribution of antibiotic resistance genes (McInnes et al., 2020), and given that Bacteroides, Lactobacilli, Clostridiales, and Coriobacteria are all common in the gut microbiome, it appears A. equolifaciens has acquired helD genes from gut microorganisms on two separate occasions. Indeed, an unusual feature of helD genes is that many organisms contain multiple paralogues and that all versions are expressed. Why some organisms have a single gene for helD while a closely related species has multiple expressed copies is unclear, and this will make a fascinating avenue for future research. It is interesting to note that actinobacteria, such as Streptomyces, Frankia, and Nonomuraea (numbers 50, 51, 54, and 55; Figure 3) that are known producers of valuable bioactive compounds used as antibiotics and anti‐cancer drugs contained the largest number of helD genes (4–5). The 5 helD genes in Nonomuraea (number 55, Figure 3), which is a known producer of DNA‐intercalating agents (Sungthong & Nakaew, 2015) may be involved in genome maintenance through recycling stalled transcription complexes during the production of these compounds. Nonomuraea and other Actinomycetales sometimes have a second rpoB gene that confers resistance of RNAP to compounds such as rifampicin and sorangicin that is induced by stress and is associated with the production of secondary metabolites (D'Argenio et al., 2016). The combination of multiple HelD isoforms with drug‐resistant RNAP may be important in this proposed genome maintenance activity. In some organisms, such as M. abcessus and S. venezuaelae helD expression is induced in the presence of the antibiotic rifampicin, conferring resistance, and this is associated with the presence of a DNA sequence called the Rifamycin Associated Element (RAE) found upstream of the gene (Hurst‐Hess et al., 2021; Surette et al., 2021). It is proposed that the tip of the PCh loop can physically remove rifampicin bound to the RNAP β subunit in a pocket close to the active site. In S. venezuelae (organism #50, Figure 3) that has five helD genes, only one (SVEN_6029, #50.3) is induced in the presence of rifampicin and has an upstream RAE (Surette et al., 2021). It is interesting to note that despite encoding a rifampicin‐resistant RNAP β subunit, Nonomuraea also has an RAE located directly upstream of helD NOA_42280 (#55.3; Appendix 1; Figures A7 and A8).

FIGURE A8

The helR group of helD variants that (potentially) confer rifampicin resistance. The phylogenetic tree from Figure 1 is shown on the left side with the region boxed expanded on the right side. Bacterial classes are colored, species numbered, the number of helD genes colored as in Figure 1. Only the (potential) helR variants are shown: 50 Streptomyces venezuelae (#3 SVEN_6029). 55 Nonomuraea sp. ATCC55076 (#3 NOA_42280, 715 aa). 58 Microbacterium sp. PAMC 28756 (mip_00070, 717aa). 59 Mirobacterium hominis SJTG1 (mhos_01135, 744aa). 60 Nocardia farcinica IFM10152 (#2 NFA_44160, 726aa). 61 Mycobacterium smegmatis MC2 155 (MSMEG_2174, 736aa). 62 Rhodococcus sp. 008 (#2 rhod_09075, 731aa). 63 Mycobacterium sp. JS623 (Mycsm_03949, 732aa). 65 Mycobacteroides abscessus ATCC 19977 (MAB_3189c, 753aa). 66 Rhodococcus equi 103S (#2 REQ_15310, 739aa). 67 Nocardia asteroides NCTC11293 (#2 nad_04408, 735aa). One helD gene, black; two, blue; five, green

Investigation of the distribution of helD genes with upstream RAEs revealed they were clustered to two sub‐branches of the Actinobacteria (Appendix 1; Figure A8) that may be considered the HelR grouping based on the nomenclature of these proteins by (Hurst‐Hess et al., 2021; Surette et al., 2021). It should be noted that clearly identifiable RAEs could not be found upstream of all the genes in the HelR group, including for Frankia alni, Nocardia brasiliensis, or Mycolicibacterium phlei (54.2, 56.2, and 64, respectively; Figure 3 and Appendix 1 Figure A2). Rifampicin has also been observed to induce helD expression in the low G+C Gram‐positive B. subtilis, but this induction does not confer resistance to the drug (Hutter et al., 2004). Nevertheless, the ability of naturally produced antibiotics to induce the expression of helD genes suggests HelD proteins have a potentially important role in preserving genome integrity and gene expression in the bacteria in which they are found.

An additional area of future research should include functional and structural studies of HelD from Gram‐negative bacteria, as due to the location of lineage‐specific inserts in the β and β’ subunits of RNAP in Gram‐negatives it was assumed HelD‐like proteins would bind poorly or be sterically inhibited from binding. HelD proteins represent a new class of motor enzymes involved in transcription complex recycling that are widely distributed in bacteria that make an important contribution to our understanding of the multiple different mechanisms used to resolve potentially lethal stalled transcription complexes.

Finally, it is important that genome annotation databases are updated as helD genes are often classified as pcrA, uvrD, or helicase IV‐ATPase. Correct annotation of helD genes will enable a more detailed understanding of the distribution, evolution, and function of this fascinating new category of transcription factors.

ETHICS STATEMENT

None required.

CONFLICT OF INTEREST

None declared.

AUTHOR CONTRIBUTIONS

Joachim S. Larsen: Formal analysis (supporting); Methodology (supporting); Software (supporting); Writing‐review & editing (supporting). Michael Miller: Formal analysis (supporting); Writing‐review & editing (supporting). Aaron J. Oakley: Formal analysis (supporting); Funding acquisition (equal); Writing‐review & editing (supporting). Nicholas E. Dixon: Formal analysis (supporting); Funding acquisition (equal); Writing‐review & editing (supporting). Peter Lewis: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Funding acquisition (equal); Investigation (lead); Methodology (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing‐original draft (lead); Writing‐review & editing (lead).

TABLE A1

comparison of conserved class I and II HelD motifs with those from class III M. xanthus HelD