Taxonomic Re-Evaluation and Genomic Comparison of Novel Extracellular Electron Uptake-Capable Rhodovulum visakhapatnamense and Rhodovulum sulfidophilum Isolates

Rhodovulum spp. are anoxygenic phototrophic purple bacteria with versatile metabolisms, including the ability to obtain electrons from minerals in their environment to drive photosynthesis, a relatively novel process called phototrophic extracellular electron uptake (pEEU). A total of 15 strains of Rhodovulum sulfidophilum were isolated from a marine estuary to observe these metabolisms in marine phototrophs. One representative strain, Rhodovulum sulfidophilum strain AB26, can perform phototrophic iron oxidation (photoferrotrophy) and couples carbon dioxide fixation to pEEU. Here, we reclassify two R. sulfidophilum isolates, strainAB26 and strain AB19, as Rhodovulum visakhapatnamense using taxonomic re-evaluation based on 16S and pufM phylogenetic analyses. The strain AB26 genome consists of 4,380,746 base-pairs, including two plasmids, and encodes 4296 predicted protein-coding genes. Strain AB26 contains 22 histidine kinases, 20 response regulators, and dedicates ~16% of its genome to transport. Transcriptomic data under aerobic, photoheterotrophy, photoautotrophy, and pEEU reveals how gene expression varies between metabolisms in a novel R. visakhapatnamense strain. Genome comparison led by transcriptomic data under pEEU reveals potential pEEU-relevant genes both unique to R. visakhapatnamense strains and shared within the R. sulfidophilum genomes. With these data we identify potential pEEU-important transcripts and how speciation may affect molecular mechanisms of pEEU in Rhodovulum species from the same environment.

Introduction
Bacterial metabolisms are diverse, allowing for growth in nearly any environment [1]. Some bacteria are capable of obtaining electrons from insoluble extracellular sources within their environments via a process called extracellular electron uptake (EEU) [2]. Various forms of this metabolism exist with the common theme being that microbes interact with solid-phase conductive substances as sources of electrons [3]. Through EEU, microbes drive biogeochemical cycles in all corners of the earth at undetermined rates [4]. This metabolism also has potential to improve bioremediation of contaminated sites and the biosynthesis of sustainable fuels [5][6][7].
EEU was first observed in species such as mineral-reducing, Shewanella oneidensis MR1 and the photoautotroph, Rhodopseudomonas palustris TIE-1 (hereafter referred to as TIE-1) [8][9][10]. One well-characterized electron uptake system is known; the PioABC operon studied in TIE-1 [11]. Studies of pEEU in TIE-1 revealed the Calvin-Benson-Bassham cycle as an electron sink during pEEU [12,13]. This link to carbon dioxide fixation connects pEEU to the carbon cycle. Furthermore, because TIE-1 can use various soluble and insoluble forms of iron, pEEU connects to the cycling of iron and other elements that are bound to iron minerals [3,14]. Until recently pEEU has been studied solely in freshwater phototrophs, leaving a knowledge gap regarding the prevalence of pEEU in other ecosystems.
In an effort to address this, we isolated bacteria from a microbial mat in a marine estuary in Woods Hole, Massachusetts, USA (henceforth referred to as Woods Hole isolates) [15,16]. In total, 15 of the Woods Hole isolates were initially identified as Rhodovulum sulfidophilum, a purple non-sulfur bacterium capable of many diverse metabolisms including anoxygenic photosynthesis (non-oxygen-evolving). We sequenced and assembled the genomes of 12 of the 15 isolates based on these data (3 isolates, namely Rhodovulum sulfidophilum strain AB14, strain AB26, AB30, were assembled previously [15]). We have previously reported that all 15 strains were capable of heterotrophic, photoautotrophic, and photoheterotrophic growth as well as photoferrotrophy [12]. A representative strain, R. sulfidophilum AB26, was selected for further growth studies and found to be capable of pEEU in bioelectrochemical systems (BES) using a novel uptake pathway consisting of a di-heme c-type cytochrome (EeuP), with carbon dioxide fixation as a cellular sink for acquired electrons [12]. EeuP is found in diverse bacterial lineages without other characterized pEEU uptake systems in their genomes, which may suggest its role as a novel uptake system [12].
In assembling and releasing the remaining 12 genomes of the Woods Hole isolates, we discovered 2 isolates, strain AB19 and the representative pEEU strain AB26, that no longer fit into R. sulfidophilum but rather the closely related, novel Rhodovulum visakhapatnamense species, originally isolated from a tidal seawater enriched under phototrophic conditions [17]. Prior to this study, R. visakhapatnamense was represented by a single-strain R. visakhapatnamense strain JA181. Like other Rhodovulum species, strain JA181 is capable of chemoorganotrophy in the dark and photoheterotrophy under illumination [17]. Currently, three R. visakhapatnamense genomes are publicly available: strain AB26, strain AB19, and strain JA181 [17]. Given this new taxonomic classification and the previously described transcriptomic data our group possesses [11], we aim to describe the genome content and expression profiles under these growth conditions to further characterize members of R. visakhapatnamense. Comparative genomic analysis based on transcript data of putative pEEU-important transcripts within R. visakhapatnamense strain AB26 reveals the distribution of homologous genes within the genomes of the 15 Woods Hole isolates.

Microbial Isolation and Cultivtion Conditions
As previously described [11], Rhodovulum sulfidophilum strains were isolated in July 2014 from independent microbial mat samples from the Trunk River estuary in Woods Hole, MA. Enrichments were cultivated photoheterotrophically in anoxic artificial seawater (SW) medium supplemented with 20 mM acetate or 10 mM sodium thiosulfate. Enrichments were cultivated with ∼850-nm light at 30 • C, and passaged six times, followed by streaking aerobically six times on Bacto agar with Difco marine broth (MB) 2216 (BD Diagnostic Systems, Hunt Valley, MD USA) to isolate single colonies. All growth experiments were carried out at 30 • C unless otherwise noted. AB26 strains were grown in MB broth under dark with shaking at 200 rpm for aerobic heterotrophic growth. All phototrophic (photoautotrophic and photoheterotrophic) growth experiments were carried out anaerobically in SW media supplemented with 70 mM sodium bicarbonate and 1 mM sodium sulfate in sealed sterile serum bottles. The phototrophic cultures were grown without shaking under light (with a single 60 W incandescent light bulb at 25 cm). For anaerobic photoautotrophic growth, AB26 strains were grown with H 2 (80% H 2 : 20% CO 2 at 50 kPa) or 10 mM sodium thiosulfate or 5 mM Fe(II) chloride in SW medium. To prevent iron precipitation, the photoautotrophic culture with Fe(II) contained 10 mM nitrilotriacetic acid (NTA). For anaerobic photoheterotrophic growth, AB26 strains were grown in SW medium with 10 mM acetate. The headspace atmosphere of the phototrophic cultures consisted of 80% N 2 and 20% CO 2 , except for hydrogen. Where a change in culture medium was required, cells were washed three times in basal SW medium post-centrifugation at 5000× g.

Genome Sequencing
Genome analysis was performed as previously described [15]. Genomic DNA was isolated from mid-log phase cell culture in marine broth using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA). Samples were prepped for Illumina 250-bp paired-end sequencing using Nextera sample prep kit (Illumina, Inc., San Diego, CA, USA), and sequenced on Illumina MiSeq platform with V2 chemistry. Reads were quality checked and adapter trimmed using Trimmomatic version 0.33 using default parameters for paired-end reads [18]. Reads were de novo assembled using CLC Genomics Workbench (CLC Bio-Qiagen, Aarhaus, Denmark), and scaffolds generated using MeDuSa [19] and R. sulfidophilum DSM 2351 as an alignment guide. Alignment of reads to R. sulfidophilum DSM 2351 was performed using Bowtie2 version 2.2.29 [20] short read mapper, and annotated with National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline [21].

Phylogenetic Analysis, Taxonomic Re-Evaluation, and Comparative Genomics
ANI between strains was performed using JSpeciesWS [22] and visualized with Morpheus (Morpheus, https://software.broadinstitute.org/morpheus (accessed on 3 February 2022)). Phylogenetic trees were built using MEGA11 [23]. For strain-level phylogenetic analysis, 16S rRNA sequences and the photosynthetic reaction center subunit M (pufM) protein sequences were used to build trees [24,25]. The 16S tree used Kimura 2-parameter model [26] and a gamma distribution of 5. The pufM tree used Jones-Taylor-Thornton (JTT) model [27] and a gamma distribution of 5. For phylogenetic analysis of EeuP protein sequences among R. sulfidophilum strains, the JTT model was used with a gamma distribution of 5. All sequences were aligned using the ClustalW algorithm. BLASTp was performed using a local protein database (Supplemental Materials File S2).

RNA Isolation, RNA Sequencing, and Differential Expression Analysis
As previously described [12], Cell cultures were sampled anaerobically and immediately mixed with 1:1 with RNAlater ® (Qiagen, Germantown, MD, USA). RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, USA) and DNA removal performed using Turbo DNA-free Kit (Ambion, Austin, TX, USA). RNA samples were tested for purity using PCR. Illumina single-end 50-bp libraries were prepared and sequenced at Washington University's Genome Technology Access Center on an Illumina HiSeq3000 (Illumina Inc., Madison, WI, USA). Reads were mapped to the strain AB26 genome using TopHat2 version 2.1.1 and the gff3 annotation file as a guide for sequence alignment. Bowtie 2 version 2.3.3.1 was used to index the reference genome FASTA file. The number of reads mapping to each feature were counted by HTSeq version 0.9.1. Differentially expressed genes were determined in DESEQ2 version 1.16.1 using the HTSeq read counts. Significantly differentially expressed genes were identified using an adjusted p-value cutoff of 0.05.

Taxonomic Re-Evaluation of Strain AB26 and Strain AB19
A total of 15 isolates were cultured from a marine microbial mat in Woods Hole, MA, USA and identified as belonging to R. sulfidophilum through 16S sequencing [12,15]. Prior to sequencing the Woods Hole isolates, few R. sulfidophilum genomes were publicly available. Whole genome alignments of strain AB26 showed only 57.8% of the genome sequence aligned to the available reference genome R. sulfidophilum DSM2351 [15]. Growth experiments of select isolates revealed differences in doubling times in the same growth conditions [12], and therefore we sought to further investigate the phylogenetic relatedness of the isolated strains. 16S and photosynthetic reaction center subunit M (pufM) phylogenetic trees were produced to determine the relatedness between isolates from Woods Hole ( Figure 1). GCA_001548075.1; IM796 GCA_016653205.1; SE1 GCA_010119435.1; SNK001 GCA_001633145.1

Taxonomic Re-Evaluation of Strain AB26 and Strain AB19
A total of 15 isolates were cultured from a marine microbial mat in Woods Hole, MA, USA and identified as belonging to R. sulfidophilum through 16S sequencing [12,15]. Prior to sequencing the Woods Hole isolates, few R. sulfidophilum genomes were publicly available. Whole genome alignments of strain AB26 showed only 57.8% of the genome sequence aligned to the available reference genome R. sulfidophilum DSM2351 [15]. Growth experiments of select isolates revealed differences in doubling times in the same growth conditions [12], and therefore we sought to further investigate the phylogenetic relatedness of the isolated strains. 16S and photosynthetic reaction center subunit M (pufM) phylogenetic trees were produced to determine the relatedness between isolates from Woods Hole ( Figure 1). Figure 1. 16S rRNA (a) and pufM (b) phylogenetic trees between Woods Hole R. sulfidophilum isolates. (a) 16S phylogenetic tree constructed by maximum-likelihood method using the Kimura-2 parameter model [26], tested by bootstrapping (500 re-samplings) in MEGA11 [22] with Pseudomonas aeruginosa as outgroup. (b) pufM tree constructed by maximum-likelihood using the Jones-Taylor-Thornton parameter model [27], tested by bootstrapping (500 re-samplings) in MEGA11 [22], using Cereibacter sphaeroides 2.4.1 as outgroup.
16S phylogenetic analysis shows strain AB26 and R. sulfidophilum strain AB19 reside in a separate branch from all other Woods Hole isolates, with a second clade consisting of strain AB33, strain AB16, and strain AB30. The remaining Woods Hole isolates generally nest within a group together. The pattern of the 16S phylogenetic tree is mirrored in the photosynthetic reaction center subunit M (pufM) tree in that strain AB26 and strain AB19 again reside in a separate branch from the remaining Woods Hole isolates. Further investigation using average nucleotide identities (ANI) revealed strain AB26 and strain AB19 ANI values are far below the cut-off value for same-species relatedness (>~94%, Figure 2) [27]. The remaining Woods Hole isolates all show ANI values indicative of same-species relatedness. Two groups of strains, AB18 and AB33 and strains AB35 and AB15, show highest similarity to each other. One other isolate, strain AB28, showed lower ANI values to all other strains (94-95% ANI) but were still within same species-values ( Figure 2). (a) 16S phylogenetic tree constructed by maximum-likelihood method using the Kimura-2 parameter model [26], tested by bootstrapping (500 re-samplings) in MEGA11 [22] with Pseudomonas aeruginosa as outgroup. (b) pufM tree constructed by maximum-likelihood using the Jones-Taylor-Thornton parameter model [27], tested by bootstrapping (500 re-samplings) in MEGA11 [22], using Cereibacter sphaeroides 2.4.1 as outgroup.
16S phylogenetic analysis shows strain AB26 and R. sulfidophilum strain AB19 reside in a separate branch from all other Woods Hole isolates, with a second clade consisting of strain AB33, strain AB16, and strain AB30. The remaining Woods Hole isolates generally nest within a group together. The pattern of the 16S phylogenetic tree is mirrored in the photosynthetic reaction center subunit M (pufM) tree in that strain AB26 and strain AB19 again reside in a separate branch from the remaining Woods Hole isolates. Further investigation using average nucleotide identities (ANI) revealed strain AB26 and strain AB19 ANI values are far below the cut-off value for same-species relatedness (>~94%, Figure 2) [27]. The remaining Woods Hole isolates all show ANI values indicative of same-species relatedness. Two groups of strains, AB18 and AB33 and strains AB35 and AB15, show highest similarity to each other. One other isolate, strain AB28, showed lower ANI values to all other strains (94-95% ANI) but were still within same species-values ( Figure 2). comparisons between strain AB26 and strain AB28 were performed to include the closely related species Rhodovulum viride and Rhodovulum visakhapatnamese (Supplemental Figure  S1) [17,28]. Strain AB28 did not show any increased similarities to other Rhodovulum genomes, suggesting the current taxonomy is correct, but may change as more Rhodovulum genomes are assembled. ANI analysis shows strain AB26 and strain AB19 share ANI values of 97.95% with R. visakhapatnamense, placing strain AB26 and strain AB19 in a new species ( Figure 3).  To determine the proper species designation of strain AB26 and strain AB19, ANI comparisons between strain AB26 and strain AB28 were performed to include the closely related species Rhodovulum viride and Rhodovulum visakhapatnamese (Supplemental Figure S1) [17,28]. Strain AB28 did not show any increased similarities to other Rhodovulum genomes, suggesting the current taxonomy is correct, but may change as more Rhodovulum genomes are assembled. ANI analysis shows strain AB26 and strain AB19 share ANI values of 97.95% with R. visakhapatnamense, placing strain AB26 and strain AB19 in a new species ( Figure 3). Microorganisms 2022, 10, x FOR PEER REVIEW 6 of 17

Strain AB26 Genome Features and Expression Analysis
Strain AB26 was chosen as a representative strain for this study due to its preferable growth characteristics [12]. The genome of strain AB26 consists of 4,380,746 bp of DNA and a GC content of 67.9%, assembled into 3 scaffolds. strain AB26 has 4375 genes, with 4296 protein-encoding genes ( Table 1). The strain AB26 genome contains genes allowing for diverse metabolic capabilities that purple non-sulfur bacteria are known for. This is in line with previous observations that strain AB26 is capable of aerobic heterotrophic growth in the dark, photoheterotrophy using succinate and acetate for carbon and electrons, and photoautotrophy, using inorganic substances, including thiosulfate and hydrogen (H2).

Strain AB26 Genome Features and Expression Analysis
Strain AB26 was chosen as a representative strain for this study due to its preferable growth characteristics [12]. The genome of strain AB26 consists of 4,380,746 bp of DNA and a GC content of 67.9%, assembled into 3 scaffolds. strain AB26 has 4375 genes, with 4296 protein-encoding genes ( Table 1). The strain AB26 genome contains genes allowing for diverse metabolic capabilities that purple non-sulfur bacteria are known for. This is in line with previous observations that strain AB26 is capable of aerobic heterotrophic growth in the dark, photoheterotrophy using succinate and acetate for carbon and electrons, and photoautotrophy, using inorganic substances, including thiosulfate and hydrogen (H 2 ).

Phototrophy
R. visakhapatnamense is a member of the purple nonsulfur bacteria capable of anoxygenic photosynthesis [29]. The genes necessary for growth via photosynthesis occur in a~50-kb region of the genome spanning four separate operons. The first operon spans a 13-kb region and encodes the reaction center, bacteriochlorophyll synthesis, carotenoid synthesis, and light-harvesting proteins. Reaction center subunits M (BV509_00330) and L (BV509_00335) share 70% identity with the reaction center genes in the model anoxygenic phototroph Cereibacter sphaeroides [30]. The second photosynthetic gene cluster (PGC) consists of tetrapyrrole biosynthesis genes bchEJ. The third PGC is homologous to the puc operon, which encodes the structural polypeptides of the light-harvesting-II peripheral antenna complex, and also contains a PucC family protein [29]. PucC encoded in model puc operons regulates response to oxygen tension and light intensity [30]. The fourth PGC spans 32-kb and contains further pigment synthesis genes, the photosynthetic reaction center subunit H, and various response regulators.
Transcriptomic data shows photosynthetic reaction center subunit M is significantly expressed during pEEU compared to aerobic growth (log 2 fold change~1; p < 0.0001), but not when grown with H 2 compared to aerobic growth (log 2 fold change~0.5; p > 0.05). The same is true for photosynthetic reaction center subunit L during pEEU compared to aerobic (log 2 fold change~1; p < 0.05), but not growth with H 2 compared to aerobic growth (log 2 fold change~0.5; p > 0.05).

Carbon Dioxide Fixation
The strain AB26 genome contains form I (BV509_05525) and form II (BV509_05520) of RuBisCO, the enzyme necessary for carbon dioxide fixation via the Calvin-Benson-Bassham (CBB) cycle [29]. The two forms reside in an operon together with the CbbQ RuBisCO-activating protein (BV509_05515), and a LsyR family transcriptional regulator (BV509_15210), which shares similarity with the RuBisCO regulator CbbR. The genes required for photorespiration via the Calvin-Benson-Bassham cycle are housed 2-Mb upstream from the RuBisCO enzyme gene cluster. As previously described [12], transcriptomic data reveals upregulation of form I RuBisCO (log 2 fold change~2, p < 0.05) but downregulation of form II RuBisCO (log 2 fold change~−0.35, p > 0.1) during pEEU, and upregulation of a formate dehydrogenase operon (log 2 fold change~4; p < 0.0001) [12]. The high upregulation of formate dehydrogenase may suggest an involvement of this enzyme in catalyzing CO 2 fixation [31].

Reducing Power
Strain AB26 is metabolically diverse, capable of aerobic heterotrophy, anaerobic photoheterotrophy, or anaerobic photoautotrophy using various inorganic compounds for electron donors (Figure 4). Strain AB26 contains a 16-kb operon coding for the synthesis and assembly of a nickel-dependent hydrogenase (BV509_06105-BV509_06195, Figure 4a). Expression of this nickel-dependent hydrogenase was highest during growth with thiosulfate compared to aerobic growth, with expression of genes within the operon ranging from 1-3.5-fold (p < 0.001, Figure 4a). Strain AB26 is also capable of growth using thiosulfate via sox-like genes organized into 3 clusters. The first cluster contains two SoxXY-like carrier proteins (BV509_04530-BV509_04535) and a single metallohydrolase SoxH (BV509_04575). The second cluster contains the canonical soxABCXYZ genes (BV509_09605-BV509_09655). soxR (BV509_15000) is 1-Mb downstream, and is a putative transcriptional regulator of the soxABCXYZ genes [32]. soxR is slightly upregulated during growth with thiosulfate compared to aerobic (log 2 fold change~1.3; p < 0.0001, Figure 4b), which corresponds with the increased expression of other genes in the sox operon. However, soxR is not upregulated during growth with acetate as compared to aerobic growth (log 2 fold changẽ −0.3; p < 0.0001), despite increased expression patterns of the other sox genes (1-2-fold; p < 0.0001, Figure 4b). A carbon monoxide dehydrogenase (BV509_12715-BV509_12730, Figure 4a) and a formate dehydrogenase operon (BV509_15145-BV509_15165, Figure 4a) are also present in the strain AB26 genome.

Biodegradation and Carbon Storage
The diverse metabolisms of purple non-sulfur phototrophs allow for proliferation in industrial waste environments [38]. Rhodopseudomonas palustris and Cereibacter sphaeroides are two purple phototrophs known for their ability to metabolize diverse compounds including fatty acids, amino acids, aromatic compounds, and lignin. They are, therefore, important species in the bioremediation of wastewater and polluted environments [39][40][41][42]. Some purple phototrophs are also capable of synthesizing carbon storage polymers such as polyhydroxalkanoates (PHA), which they degrade in carbon-starved environments. The genome of strain AB26 encodes a variety of genes for the metabolism of aromatic compounds: a benzoate transporter (BV509_16875) and a 4-hydroxybenzoate octaprenyltransferase (BV509_17190) for the first step of benzoate degradation, a salicylate esterase (BV509_RS17105), a biphenyl-2,3-diol 1,2-dioxygenase III-related protein (BV509_11920) for the degradation of biphenyl, a type II 3-dehydroquinate dehydratase (BV509_13850) for the degradation of quinate, and for the degradation of gentisate, a fumarylacetoacetate hydrolase family protein (BV509_03940) and a maleylacetoacetate isomerase (BV509_16060). Strain AB26 contains two operons for the degradation of phenylacetate (BV509_03065-BV509_03095, BV509_05275-BV509_05285) [43]. Similar to R. palustris TIE-1 [42], strain AB26 encodes genes for the synthesis and catabolism of polyhydroxybutyrate (PHB). phaA, which encodes a beta-ketothiolase, and phaB, which encodes an acetoacetyl-CoA reductase, are organized separately from each other and the remaining PHB gene cluster. The remainder of the PHB cluster contains the PHA depolymerase gene phaZ, the class 1 poly(R)-hydroxyalkanoic acid synthase phaC, which encodes the phasin protein affecting PHB granule accumulation and utilization, and the PHA synthesis repressor gene phaR (BV509_06270-BV509_06285). Unlike TIE-1, strain AB26 only has single-gene copies for the synthesis and utilization of PHB. As previously described [12], the PHB biosynthesis enzymes were downregulated during pEEU as compared to aerobic growth (log2fold change ~0, p > 0.05), and the repressor phaR was slightly upregulated (log2fold change ~1, p < 0.01).

Biodegradation and Carbon Storage
The diverse metabolisms of purple non-sulfur phototrophs allow for proliferation in industrial waste environments [38]. Rhodopseudomonas palustris and Cereibacter sphaeroides are two purple phototrophs known for their ability to metabolize diverse compounds including fatty acids, amino acids, aromatic compounds, and lignin. They are, therefore, important species in the bioremediation of wastewater and polluted environments [39][40][41][42]. Some purple phototrophs are also capable of synthesizing carbon storage polymers such as polyhydroxalkanoates (PHA), which they degrade in carbon-starved environments. The genome of strain AB26 encodes a variety of genes for the metabolism of aromatic compounds: a benzoate transporter (BV509_16875) and a 4-hydroxybenzoate octaprenyltransferase (BV509_17190) for the first step of benzoate degradation, a salicylate esterase (BV509_RS17105), a biphenyl-2,3-diol 1,2-dioxygenase III-related protein (BV509_11920) for the degradation of biphenyl, a type II 3-dehydroquinate dehydratase (BV509_13850) for the degradation of quinate, and for the degradation of gentisate, a fumarylacetoacetate hydrolase family protein (BV509_03940) and a maleylacetoacetate isomerase (BV509_16060). Strain AB26 contains two operons for the degradation of phenylacetate (BV509_03065-BV509_03095, BV509_05275-BV509_05285) [43]. Similar to R. palustris TIE-1 [42], strain AB26 encodes genes for the synthesis and catabolism of polyhydroxybutyrate (PHB). phaA, which encodes a beta-ketothiolase, and phaB, which encodes an acetoacetyl-CoA reductase, are organized separately from each other and the remaining PHB gene cluster. The remainder of the PHB cluster contains the PHA depolymerase gene phaZ, the class 1 poly(R)-hydroxyalkanoic acid synthase phaC, which encodes the phasin protein affecting PHB granule accumulation and utilization, and the PHA synthesis repressor gene phaR (BV509_06270-BV509_06285). Unlike TIE-1, strain AB26 only has single-gene copies for the synthesis and utilization of PHB. As previously described [12], the PHB biosynthesis enzymes were downregulated during pEEU as compared to aerobic growth (log 2 fold changẽ 0, p > 0.05), and the repressor phaR was slightly upregulated (log 2 fold change~1, p < 0.01).

Transporters
The strain AB26 genome encodes 561 transport system genes, dedicating~16% of its genome to transport. Of these, 347 transport genes are involved in primary transport, with 45 different ABC transport systems and 23 ATPase genes. Strain AB26 contains type III secretion system genes for flagella biosynthesis, and seven type II secretion system families ( Figure 6). The lack of siderophore biosynthesis genes suggests that strain AB26 may transport siderophores produced by other bacteria into the cell. Strain AB26 contains two ferrous iron transport genes (BV509_02645-BV509_02650) for aerobic iron acquisition. Nutrient acquisition ( Figure 6) for strain AB26 includes urea transport (BV509_09920-BV509_09945) and sulfate transport (BV509_13630-13645). Located on a plasmid are various metal uptake systems [15] including zinc (BV509_08850, BV509_19470, BV509_19480, BV509_20850), nickel (BV509_20695-BV509_20715, BV509_11055), and manganese (BV509_03550, BV509_20820-20825, Figure 6), which may help maintain metal ion homeostasis. Strain AB26 contains four microcin transporters, 14 multidrug transporters, and 9 efflux transporters. The Sec pathway was upregulated compared to aerobic growth only during pEEU, supporting the requirement of protein export in the periplasmic space to facilitate extracellular electron transfer across the membranes (Figure 6) Strain AB26 contains 176 passive transport genes, with two multidrug and toxic compound extrusion (MATE) genes, 10 resistance-nodulation-cell division (RND) pumps, 23 major facilitator superfamily (MFS) genes, and 59 tripartite ATP-independent periplasmic (TRAP) transport genes. The TRAP family transporters are common in marine bacteria such as strain AB26 due to the natural Na + gradient in their environment [44].

Nitrogen Assimilation and Fixation
Strain AB26 uses the glutamine synthetase and glutamate synthase for ammonia assimilation in the cell. Strain AB26 genome contains two ammonia transporters, three glutamine synthetases, and a glutamine-synthetase adenyltransferase to regulate activity of glutamine-synthetases. Strain AB26 also encodes genes to convert urea to ammonia In total, 60 of the ABC transport genes were identified as various amino acid transport systems, with 22 genes dedicated to the transport of branched chain amino acids. For iron acquisition, strain AB26 contains 11 tonB genes and 5 iron transport systems. The lack of siderophore biosynthesis genes suggests that strain AB26 may transport siderophores produced by other bacteria into the cell. Strain AB26 contains two ferrous iron transport genes (BV509_02645-BV509_02650) for aerobic iron acquisition. Nutrient acquisition ( Figure 6) for strain AB26 includes urea transport (BV509_09920-BV509_09945) and sulfate transport (BV509_13630-13645). Located on a plasmid are various metal uptake systems [15] including zinc (BV509_08850, BV509_19470, BV509_19480, BV509_20850), nickel (BV509_20695-BV509_20715, BV509_11055), and manganese (BV509_03550, BV509_20820-20825, Figure 6), which may help maintain metal ion homeostasis. Strain AB26 contains four microcin transporters, 14 multidrug transporters, and 9 efflux transporters. The Sec pathway was upregulated compared to aerobic growth only during pEEU, supporting the requirement of protein export in the periplasmic space to facilitate extracellular electron transfer across the membranes (Figure 6) Strain AB26 contains 176 passive transport genes, with two multidrug and toxic compound extrusion (MATE) genes, 10 resistance-nodulation-cell division (RND) pumps, 23 major facilitator superfamily (MFS) genes, and 59 tripartite ATP-independent periplasmic (TRAP) transport genes. The TRAP family transporters are common in marine bacteria such as strain AB26 due to the natural Na + gradient in their environment [44].

Extracellular Electron Uptake (EEU) Genes
To perform pEEU, Gram-negative bacteria require uptake systems consisting of electron transfer proteins spanning the outer membrane, periplasmic space, and inner membrane. These systems may also include porin proteins to span membranes and facilitate the exchange of electrons from the cell exterior to the interior. While some characterized electron uptake systems exist [11,45], these uptake systems or homologs of them are not present in all bacteria that are hypothesized to be able to perform pEEU. Our group

Extracellular Electron Uptake (EEU) Genes
To perform pEEU, Gram-negative bacteria require uptake systems consisting of electron transfer proteins spanning the outer membrane, periplasmic space, and inner membrane. These systems may also include porin proteins to span membranes and facilitate the exchange of electrons from the cell exterior to the interior. While some characterized electron uptake systems exist [11,45], these uptake systems or homologs of them are not present in all bacteria that are hypothesized to be able to perform pEEU. Our group recently identified a novel protein important to phototrophic extracellular electron uptake in strain AB26, which does not encode any known EEU pathways in its genome; the di-heme c-type cytochrome EeuP [12]. As previously reported, EeuP is a novel pEEU-important protein found in A26 but also in species spanning the Proteobacteria and Acidobacteria. So far, no other components of the novel EEU pathway have been identified in strain AB26 other than EeuP. To identify other potential proteins that might be important in pEEU, a RNASeq guided comparative genomic approach with specific targets, such as cytochrome c-containing proteins, would be useful. This is because these proteins are often associated with extracellular electron transfer [3].

RNA-Seq Led Genomic Comparison between Rhodovulum spp.
Our group previously performed RNA-Seq in strain AB26 between photoautotrophy with H 2 /CO 2 as electron donor/carbon source compared to pEEU via poised electrodes [12] in an effort to characterize the molecular mechanisms of pEEU in this novel isolate and identify the cellular sinks of electrons derived from extracellular sources. With these data, and the full genome sequences of the 14 other isolates comprising two Rhodovulum species, we can identify homologs of pEEU-important transcripts and infer ability to perform pEEU based on their presence or absence in each strain. Supplementary Table S1 shows the general genome features of all 15 Woods Hole isolates and publicly available genomes for both R. visakhapatnamense [46] and R. sulfidophilum DSM 2351 [47] and DSM1374 [48].

C-Type Cytochromes (Cyt c )
Electron transfer with solid phase donors is commonly carried out by c-type cytochromes. c-type cytochromes mediate single electron transfers via a covalently bonded heme group [49,50], making them ideal candidates for pEEU processes. Therefore, our analysis of the pEEU vs. hydrogen transcript data focuses on identifying upregulated transcripts encoding c-type cytochromes, or upregulated hypothetical proteins and searching for the canonical CXXCH (where X is any amino acid residue) heme-binding motif within the protein sequence.
Diheme Cyt c EeuP RNA-Seq identified a significantly upregulated transcript (log 2 fold change~2, p < 0.0001) in strain AB26 encoding the hypothetical protein later identified to be the di-heme EeuP (BV509_10070). BLASTp analysis identified homologous sequences of EeuP in all isolates except strain AB21. BLASTp results for strain AB21 returned two partial cyt c sequences and one partial hypothetical protein sequence. These partial sequences separately align with the intact EeuP homologs and may be an artifact of assembly. From this we can hypothesize that all the isolates are pEEU-capable. Maximum likelihood phylogenetic analysis of the EeuP protein sequence (Figure 8) reveals EeuP in the isolates reflects the same relationship shown in the 16S and pufM phylogenetic trees (Figure 1), in that strain AB19 and strain AB26 EeuP sequences group outside of the R. sulfidophilum EeuP sequences. As previously reported [12], EeuP homologous sequences were also identified in Alpha-, Beta-, and Gammaproteobacteria classes and in one Acidobacter species. Further analysis of the species containing EeuP homologs may reveal the relationship between EeuP conservation and metabolic/environmental adaptation.

Hypothetical Protein BV509_18570
Another significantly upregulated transcript (log2fold change ~6, p < 0.0001) in strain AB26 encodes a hypothetical protein (BV509_18570). BV509_18570 is a 413-residue long protein containing a single heme-binding motif (CXXCH). InterProScan [51] predicts the molecular functions as electron transfer and heme binding. PRED-TAT [52] did not predict a secretory signal peptide (Sec), which would be expected for an electron transport protein involved in trans-membrane transport. Surprisingly, BLASTp analysis revealed no homologs of this open reading frame (ORF) within any of the R. sulfidophilum isolates. Homologs of BV509_18570 were found in R. visakhapatnamense strain AB19 (JMJ92_02255) and R. visakhapatnamense strain JA181 (EV657_1244). The homologous sequences are each 625 amino acid residues in length, ~200 amino acid residues longer than the strain AB26 BV509_18570. Protein sequence alignments in MAUVE [53] show JMJ92_02255 and EV657_1244 sequences align 212 residues before aligning to BV509_18570, and these additional residues contain a second heme-binding motif and a Sec signal peptide sequence. Therefore, it is possible the 413-residue long BV509_18570 may be truncated due to an assembly error and, similar to the related homologs, contains an additional heme-binding motif and Sec signal peptide sequence. BV509_18570 could be a another di-heme cytc similar to EeuP, which was also annotated as a hypothetical protein, and be important for pEEU specific to R. visakhapatnamense spp. This hypothesis is supported by the high log2fold change of this transcript during pEEU and presence of a Sec signal peptide sequence, suggesting the transport of this protein across the inner membrane to the periplasmic space.

Hypothetical Protein BV509_18570
Another significantly upregulated transcript (log 2 fold change~6, p < 0.0001) in strain AB26 encodes a hypothetical protein (BV509_18570). BV509_18570 is a 413-residue long protein containing a single heme-binding motif (CXXCH). InterProScan [51] predicts the molecular functions as electron transfer and heme binding. PRED-TAT [52] did not predict a secretory signal peptide (Sec), which would be expected for an electron transport protein involved in trans-membrane transport. Surprisingly, BLASTp analysis revealed no homologs of this open reading frame (ORF) within any of the R. sulfidophilum isolates. Homologs of BV509_18570 were found in R. visakhapatnamense strain AB19 (JMJ92_02255) and R. visakhapatnamense strain JA181 (EV657_1244). The homologous sequences are each 625 amino acid residues in length,~200 amino acid residues longer than the strain AB26 BV509_18570. Protein sequence alignments in MAUVE [53] show JMJ92_02255 and EV657_1244 sequences align 212 residues before aligning to BV509_18570, and these additional residues contain a second heme-binding motif and a Sec signal peptide sequence. Therefore, it is possible the 413-residue long BV509_18570 may be truncated due to an assembly error and, similar to the related homologs, contains an additional heme-binding motif and Sec signal peptide sequence. BV509_18570 could be a another di-heme cyt c similar to EeuP, which was also annotated as a hypothetical protein, and be important for pEEU specific to R. visakhapatnamense spp. This hypothesis is supported by the high log 2 fold change of this transcript during pEEU and presence of a Sec signal peptide sequence, suggesting the transport of this protein across the inner membrane to the periplasmic space.

Diheme Cyt c BV509_14915
This transcript (log 2 fold change~6, p < 0.0001) encodes a cyt c with two heme-binding motifs. BV509_14915 is 347 residues long and contains a Sec signal peptide. Because of this, BLASTp analysis shows all 15 marine Rhodovulum spp. isolates contain a homolog of this di-heme cyt c . Given the high log 2 fold change, Sec signal peptide sequence, heme-binding motifs, and the presence of homologs within the other isolates' genomes, this cytochrome could be a conserved component of the pEEU molecular pathway in the Rhodovulum spp. isolates.

Cyt c BV509_15885
The transcript encoding the gene BV509_15885 is annotated as a cyt c . RNA-Seq data did not identify this cyt c as significantly upregulated during pEEU (log 2 fold change −0.48), but it was identified through heme staining (membrane fraction) followed by mass spectroscopy compared to hydrogen grown cultures [12]. This cyt c contains 5 heme-binding motifs and a predicted Sec signal peptide. BLASTp analysis identified homologs of this cyt c in strain AB19 and R. visakhapatnamense strain JA181. Homologs in the R. sulfidophilum isolates show 71-75% identity.

Transcriptional Regulators
To understand expression patterns and potential regulators of pEEU genes, the pEEU vs. hydrogen transcript data was analyzed for any upregulated transcriptional regulators.

Mar Family Transcription Regulator BV509_01095
BV509_01095 encodes a hypothetical protein identified as a member of the multi antibiotic resistance family transcriptional regulator (log 2 fold change~6, p < 0.001). InterProScan predicts phosphorelay signal transduction function. BV509_01095 is present only in strain AB19 and strain AB26 via BLASTp analysis, but not R. visakhapatnamense strain JA181 or any of the R. sulfidophilum isolates.

Transcriptional Regulator BV509_15825
This transcript is another transcriptional regulator upregulated during pEEU in strain AB26 (log 2 fold change~4, p < 0.0001). This regulator is also predicted to be involved in signal transduction response regulation via InterProScan, with DNA-binding wing-helix domain and tetratricopeptide repeat domain. BLASTp reveals this transcription regulator is unique to strain AB19, strain AB26, and R. visakhapatnamense strain JA181 but not the R. sulfidophilum isolates.

Conclusions
With the current accessibility of genome sequencing and ability to isolate bacterial species from diverse environments, this paper shows that taxonomy requires continuous addressing. Here, we provide a current taxonomic classification of 15 Rhodovulum spp. isolated from a marine estuary, specifically the re-classification of two of the original 15 isolates previously identified as R. sulfidophilum now as R. visakhapatnamense. These isolates are capable of many modes of metabolism, including photoferrotrophy and pEEU. To expand the characterization of R. visakhapatnamense, which at present is represented by 3 genomes assembled at the scaffold level, we describe the genome contents of R. visakhapatnamense strain AB26, and pair the genome data with expression data under anaerobic photoheterotrophy (acetate), photoautotrophy (H 2 or thiosulfate as electron donor), and photosynthetic extracellular electron uptake (poised electrode as electron donor). Upregulated transcripts under pEEU of interest to the analysis include c-type cytochromes and transcriptional regulators. BLASTp shows some transcripts of interest are unique to R. visakhapatnamense genomes and could be indicative of adaptation of molecular mechanisms for pEEU explained by speciation.
What will be interesting to observe in the future is continued comparison of expression profiles and proteomes under pEEU and photoferrotrophy between R. sulfidophilum and R. visakhapatnamense strains from Woods Hole, as this data points to differences between species. Mutational analysis of genes of interest in each species will be necessary to determine their role under these growth conditions. With the data presented here, we can begin to characterize the metabolic pathway of pEEU in Rhodovolum species.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms10061235/s1, File S1: database files for phylogenetic trees ( Figure S1: Average Nucleotide Identity (ANI) heatmap comparing AB26, AB19, and AB28 to other closely related Rhodovulum strains. The heatmap shows AB19 and AB26 share >99% ANI to each other, and 98% ANI to Rhodovulum visakhapatnamense. AB28 does not show increased ANI values to any related species, despite the relatively low (~94%) ANI to the other Woods Hole isolates ( Figure 2). ANI values were calculated using JSpeciesWSd and visualized as a heatmap using Morpheus. Table S1: General Genome Features of Woods Hole isolates and publicly available Rhodovulum visakhapatnamense and Rhodovulum sulfidophilum genomes from NCBI and IMG/JGI databases); File S2: RNASeq Heatmaps base files.


Anatomy of Omicron neutralizing antibodies in COVID-19 mRNA vaccinees

 SARS-CoV-2 vaccines, administered to billions of people worldwide, are mitigating the effects of the COVID-19 pandemic, however little is known about the molecular basis of antibody cross-protection to emerging variants, such as Omicron (B.1.1.529), and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine1, were tested for neutralization against the Omicron variant and SARS-CoV-1 virus. Cross-neutralizing antibodies were isolated from 100% of seropositive and 20% of seronegative vaccinees. Only 14.2% and 4.0% of tested antibodies neutralized the Omicron variant and SARS-CoV-1 respectively. These nAbs recognized mainly the SARS-CoV-2 receptor binding domain (RBD) and targeted class 3 and class 4 epitope regions on the SARS-CoV-2 spike protein. Antibodies targeting class 1/2 epitope regions only rarely showed cross-neutralization activity. Cross-protective antibodies derived from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1 and IGHV1-69;IGHV4-1. Only 15.6% and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralized Omicron and SARS-CoV-1 respectively. Our data provide evidence of the presence of cross-neutralizing antibodies induced by vaccination and map conserved epitopes on the S protein that can inform vaccine design.

Introduction
Since its rst appearance in December 2019, more than 370 million cases of SARS-CoV-2 infections were reported worldwide, with over 5.6 million deaths. Effective vaccines against the virus that rst appeared in Wuhan, China, have been developed with unprecedented speed. However, their ability to contain the global pandemic has been compromised by the inability to timely deliver vaccines to low-income countries and by the appearance of several antigenic variants which escaped the natural and vaccineinduced immunity [2][3][4] . The main variants that emerged so far, and listed as variants of concern (VoCs), are named B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) and B.1.1.529 (Omicron) 5,6 .
The latter one showed to be the most e cient in spreading into partially immune populations and in few months from its appearance have conquered most regions of the world 7,8 . The Omicron variant contains 37 mutations in the spike (S) protein, with up to 15 mutations in the receptor binding domain (RBD), the primary target for neutralizing antibodies (nAbs). Several reports have shown that the unprecedented number of mutations carried on the Omicron S protein drastically reduce up to 40-fold the neutralizing e cacy of sera from infected and vaccinated people and that this VoC can escape more than 85% of nAbs described in literature, including several antibodies approved for clinical use by regulatory agencies [9][10][11][12][13][14][15][16][17] . While serum activity and neutralization e cacy of selected mAbs against Omicron have been extensively reported, the functional and genetic anatomy of nAbs elicited in naïve (seronegative) and convalescent (seropositive) people immunized with the BNT162b2 mRNA vaccine remains to be explored. Taking advantage of our previous work 1 , we tested 276 human monoclonal antibodies able to neutralize the original SARS-CoV-2 virus isolated in Wuhan, for their ability to neutralize the Omicron variant and the distantly related SARS-CoV-1 virus. Our work unravels the genetic signature of cross-protective antibodies and mapped conserved sites of pathogen vulnerability on the S protein that can be used to design the next generation of COVID-19 vaccines.

Omicron effects on vaccine-induced nAbs
To understand the impact of SARS-CoV-2 Omicron on the antibody response, we evaluated the neutralization activity of 276 nAbs previously isolated from seronegative (n=52) and seropositive (n=224) donors immunized with the BNT162b2 mRNA vaccine (Fig. 2) 1 . While Omicron cross-neutralizing nAbs were identi ed in all seropositives, these antibodies were found only in one out of ve seronegatives (20%) (Extended Data Table 1). Only 1 out of 52 nAbs from seronegatives (1.9%) neutralized Omicron with a medium-low neutralization potency ( Fig. 2a and c). Since only 1 nAb was identi ed from this group no statistical difference in the geometric mean (GM) 100% inhibitory concentration (IC 100 ) between the Wuhan and Omicron viruses was found in this group. Conversely, 38 out 224 nAbs from seropositives (16.9%) were able to neutralize Omicron. These nAbs showed a 3.16-fold decreased neutralization potency compared to the Wuhan virus showing a GM-IC 100 of 719.8 ng/mL (Fig. 2b-c). Overall, 39 Omicron nAbs were identi ed of which 38 (97.4%) targeted the S protein RBD and 1 (1.6%) recognized the NTD (Extended Data Fig. 2b, left bar). None of the 39 identi ed nAbs showed an extreme neutralization potency (IC 100 below 10 ng/mL) against Omicron. To identify immunodominant sites on the Omicron S protein a ow cytometry-based competition assay was performed. In our previous study, we found that 215/276 (77.9%) nAbs bound to the S protein RBD and the majority of these antibodies were competing with J08, which epitope spans between Class 1 and Class 2 regions, and S309, which target the Class 3 region 1 . In this work all 215 RBD targeting-nAbs were additionally tested by competition assay against Class 4 targeting mAb CR3022. Class 1/2, Class 3 and Not-competing nAbs were found in both seronegatives and seropositives, while Class 4 competing nAbs were found exclusively in seropositives (Extended Data Fig. 2a; Extended Data Table 2). In both groups, Class1/2 competing nAbs were the most abundant constituting 70.3% (n=26) and 64.0% (m=114) of all antibodies isolated from seronegatives and seropositives respectively (Extended Data Fig. 2a; Extended Data Table 2). The second most abundant group of mAbs were Not-competing (n=4; 10.8%) and Class 3-competing nAbs (n=47; 27.0%) for seronegatives and seropositives respectively. Finally, only 7 (3.4%) Class 4 competing nAbs were found exclusively in seropositives (Extended Data Fig. 2a; Extended Data Table 2). From the 215 tested nAbs, we found that only 20 out of 140 (14%) Class 1/2 targeting nAbs were able to neutralize Omicron, while up to 29% percent (15/51) of Class 3 nAbs showed neutralization activity against this variant ( Fig. 2d-e). Finally, 14% (1/7) Class 4-competing nAbs were able to neutralize the Omicron virus (Fig. 2f). Among the 39 RBD-targeting Omicron nAbs, the majority recognized the Class 1/2 region (n=20; 51.3%) followed by Class 3 (n=15; 38.5%), while nAbs targeting Class 4 region (n=2; 5.1%) and Not-competing Antibody cross-protection to SARS-CoV-1 We investigated the ability of COVID-19 mRNA vaccine elicited nAbs to neutralize the ancestral SARS-CoV-1 (SARS1) virus, using lentiviral vector derived pseudoparticles, to identify immunodominant sites of cross-protection across different coronaviruses 25 . All 276 previously identi ed nAbs were tested for their ability to bind the SARS1 S protein (Extended Data Fig. 1). Four out of ve seropositives (80%) and one out of ve seronegatives (20%) showed nAbs able to bind the SARS1 S protein ( Fig. 3a; Extended Data Table 1). Of the 52 nAbs isolated from seronegatives 3 (7.5%) recognized SARS1 S protein, while 16 out of 224 nAbs (14.8%) from seropositives were able to bind this antigen ( Figure 3a). When nAbs were tested for their neutralization activity against SARS1, only 1 (1.9%) and 10 (4.5%) nAbs where found from seronegatives and seropositives respectively (Fig. 3b). All SARS1 cross-neutralizing nAbs recognized the SARS-CoV-2 S protein RBD (Extended Data Fig. 2b, right bar). Class 4 targeting nAbs able to crossneutralize SARS1 were overall the most frequent, followed by Class 3 binding nAbs (Fig. 3c). While Class 4 were the most frequent, Class 3 targeting nAbs showed the highest neutralization potency (Fig. 3c-d).

Discussion
In this work we deeply characterized an extensive panel of vaccine elicited-neutralizing human monoclonal antibodies against the heavily mutated Omicron variant and the distantly related SARS-CoV-1 virus to understand the degree of cross-protection and to map conserved regions on the S protein. We found that only 14.2% and 4.0% of our antibody panel was able to neutralize the Omicron VoC and the SARS-CoV-1 virus respectively. Remarkably, from the group of seronegative people vaccinated with two doses of the BNT162b2 mRNA vaccine, we isolated only 1 of the 39 nAbs against Omicron while none of their antibodies was able to neutralize SARS-CoV-1. Cross-neutralizing antibodies were isolated almost exclusively from people that were vaccinated after infection highlighting once again the broad crossprotection conferred by hybrid immunity 1,26 . Despite these results, we observed that antibody germlines mainly involved in Omicron and SARS-CoV-1 cross-protection (IGHV1-58;IGHJ3-1 and IGHV1-69;IGHJ4-1) were used in both seronegative and seropositive vaccinees suggesting that a third booster dose in naïve people could enhance germline-maturation and induce a more broad and persistent antibody response 27 . Furthermore, a third booster dose could drive a nity maturation of poorly cross-reactive but predominant RBD-targeting B cell germlines elicited following SARS-CoV-2 Wuhan infection or vaccination, which were shown to persist for up to 6 months in the draining lymph nodes of vaccinated individuals 28 . These data suggest that primary immunization with two doses of vaccine in naïve people is not su cient to elicit a meaningful proportion of cross-neutralizing antibodies, and that this requires a secondary immune response that can be obtained by vaccinating previously infected people or by providing a third booster dose 29 . Further studies will be necessary to understand whether a booster dose in naïve people can elicit a hybrid immunity-like antibody response and to de ne the molecular basis of cross-protection in this population. In addition to this observation, we herein de ned the epitope regions that mediate crossprotection with Omicron and SARS-CoV-1. Indeed, we observed that, while the majority of nAbs against SARS-CoV-2 Wuhan virus recognize the tip of the receptor binding domain, which comprise antigenic Classes 1 and 2, the larger proportion of cross-neutralizing antibodies map in the lower portion of the RBD which comprises exclusively antigenic Classes 3 and 4. This information can support the design of next-generation COVID-19 vaccines broadly protective against current and future variants. containing a sub-con uent Vero E6 cell monolayer. Plates were incubated for 3-4 days at 37°C in a humidi ed environment with 5% CO 2 , then examined for CPE by means of an inverted optical microscope by two independent operators. All nAbs were tested at a starting dilution of 1:8, diluted step 1:2, and the IC 100 evaluated based on their initial concentration. Technical duplicates for each experiment were performed. In each plate positive and negative control were used as previously described 1,20 .

SARS-CoV-2 virus variants CPE-MN neutralization assay
The SARS-CoV-2 Omicron (B.1.1.529) virus used to perform the CPE-MN neutralization assay was supplied and sequenced by the NRC UZ/KU Leuven (Leuven, Belgium). Sequence was deposited on GISAID with the following ID: EPI_ISL_6794907.

SARS-CoV-2 S protein competition assay
Competitive Flow cytometry-based assay was performed to characterize nAbs binding pro les to SARS-CoV-2 S-protein as previously described 1 . Brie y, magnetic beads (Dynabeads His-Tag, Invitrogen) were covered with His-tagged S-proteins, following manufacturers' instructions. Then, 40 mg/mL of beadsbound-S-protein were incubated with unlabeled nAbs for 40 minutes at RT. Following incubation, samples were washed with PBS and incubated with uorescently labeled Class 1/2 (J08-A647), Class 3 (S309-A488) or Class 4 (CR3022-A647) S-protein nAbs binders. Antibodies labelling was performed using Alexa Fluor NHS Ester kit (Thermo Scienti c). Following 40 minutes of incubation at RT, beads-antibodies mix was washed with PBS, resuspended in 150 μL of PBS-BSA 1% and acquired using BD LSR II ow cytometer (Becton Dickinson). Results were analyzed using FlowJo™ Software (version 10). Beads with or without S-protein incubated with labeled antibodies were used as positive and negative controls respectively.

SARS-CoV-1 neutralization assay
For neutralization assay, HEK293TN-hACE2 cells were plated in white 96-well plates in complete DMEM medium. 24h later, cells were infected with 0.1 MOI of SARS-CoV-1 pseudoparticles that were previously incubated with serial dilution of puri ed or not puri ed (cell supernatant) mAb . In particular, a 7-point dose-response curve (plus PBS as untreated control), was obtained by diluting mAb or supernatant respectively ve-fold and three-fold. Thereafter, nAbs of each dose-response curve point was added to the medium containing SARS-CoV-1 pseudoparticles adjusted to contain 0.1 MOI. After incubation for 1h at 37°C, 50 µl of mAb/SARS-CoV-1 pseudoparticles mixture was added to each well and plates were incubated for 24h at 37°C. Each point was assayed in technical triplicates. After 24h of incubation cell infection was measured by luciferase assay using Bright-Glo™ Luciferase System (Promega) and In nite F200 plate reader (Tecan) was used to read luminescence. Obtained relative light units (RLUs) were normalized to controls and dose response curve were generated by nonlinear regression curve tting with GraphPad Prism to calculate Neutralization Dose 50 (ND 50 ).
Functional repertoire analyses nAbs VH and VL sequence reads were manually curated and retrieved using CLC sequence viewer (Qiagen). Aberrant sequences were removed from the data set. Analyzed reads were saved in FASTA format and the repertoire analyses was performed using Cloanalyst     Functional characterization of SARS1 nAbs. a, The bar graph shows the percentage of not-binding antibodies (grey), SARS1-binding nAbs from individuals who were seronegative (dark blue) and SARS1binding nAbs for individuals who were seropositive (dark red). The total number (n) of antibodies tested per individual is shown on the top of each bar. b, Dot chart shows the neutralization potency, reported as ND 50 (ng ml −1 ), of nAbs isolated from seronegatives and seropositives against SARS1. The number and percentage of nAbs from individuals who were seronegative and seropositive and neutralization ND 50 (ng ml −1 ) ranges (black dotted lines) are denoted on the graph. c-d, dot chart shows Class 1/2, Class 3 and Class4 distribution of SARS1 nAbs (c). Neutralization potencies, reported as ND 50 (ng ml −1 ), and geometric mean (black line) are denoted on the graph. The table shows the ND 50 (ng ml −1 ) geometric mean (GM) of all nAbs pulled together from each group against SARS1. Technical triplicates were performed for each experiment (d). Genetic characterization of Omicron and SARS1 nAbs. a, The graph shows the IGHV-J rearrangement frequencies between vaccinees who were seronegative (SN) and seropositive (SP) (top), and the frequency within Omicron (ο) (middle) and SARS1 (S1) (bottom) nAbs. b-i, The graphs show the neutralization potency (IC 100 ) of IGHV1-2;IGHJ6-1 (b), IGHV1-58;IGHJ3-1 (c), IGHV1-69;IGHJ4-1 (d), IGHV3-30;IGHJ6-1 (e), IGHV3-53;IGHJ6-1 (f), IGHV3-66;IGHJ4-1 (g) and IGHV2-5;IGHJ4-1 (h) gene derived nAbs, against the original SARS-CoV-2 virus rst detected in Wuhan and the B.1.1.529 (Omicron) VoC. The graph shows the neutralization potency (ND 50 ) of predominant gene derived nAbs against SARS1 (i).
Numbers and percentages of nAbs tested for each germline and neutralization ranges (black dotted lines) are denoted on each graph. j-k, violin plots show differences in the aminoacidic (aa) CDRH3 length and percentage of V gene somatic mutations for all IGHV1-58;IGHJ3-1 (j) and IGHV1-69;IGHJ4-1 (k) compared to Omicron and SARS1 nAbs respectively.

Supplementary Files
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Presence and function of Hbl B’, the fourth protein component encoded by the hbl operon in Bacillus cereus

ABSTRACT The genes hblC, hblD and hblA encode the components Hbl L2, L1 and B of the pore forming enterotoxin haemolysin BL of Bacillus cereus. Two variants of the operon existand the more common one additionally contains hblB downstream of hblCDA. Up to now, it was completely unclear whether the corresponding protein, Hbl B’, is widely expressed among B. cereus strains and if it has a distinct function. In the present study, it was shown that the hblB gene is indeed expressed and the Hbl B’ protein is secreted by nearly all analysed B. cereus strains. For the latter, a detection system was developed based on monoclonal antibody 11A5. Further, a distinct reduction of cytotoxic and haemolytic activity was observed when recombinant (r)Hbl B’ was applied simultaneously with L2, L1 and B. This effect was due to direct interaction of rHbl B’ with L1. D−6B. cereusAltogether, we present the first simple tool for the detection of Hbl B’ in B. cereus culture supernatants. Moreover, an important regulatory function of Hbl B’ in the mechanism of Hbl was determined, which is best described as an additional control of complex formation, balancing the amounts of Hbl B-L1 complexes and the corresponding free subunits.

Introduction
Hemolysin BL is a primary virulence factor produced by members of the Bacillus cereus group. It is involved in endophthalmitis [1,2] and causes fluid accumulation in ligated rabbit ileal loops with an activity comparable to cholera toxin [3]. Furthermore, it is dermonecrotic [4], hemolytic toward erythrocytes from several species [4][5][6], active in vascular permeability tests [4] and it shows cytotoxicity to a variety of different cell lines [3,[7][8][9]. Hbl was first identified as a binary bacterial toxin consisting of a binding (B) and a lytic (L) component [5], then the three components L2, L1, and B were described [10]. In these early studies, the characteristic ring-shaped discontinuous hemolysis pattern on blood agar plates was discovered. Hemolysis occurs at an appropriate concentration ratio of Hbl L2, L1, and B [3,10,11]. Newer studies using purified recombinant (r)Hbl components and B. cereus culture supernatants showed that hemolysis depends on concentration and diffusion velocity of Hbl B. While rHbl L2 does not seem to have an impact on the outer hemolysis ring, it is enhanced by additional rHbl B and hindered or disrupted by additional rHbl L1 [12].
Hbl is a pore forming toxin, which was first shown by osmotic protection assays [11]. The Hbl components are structurally very similar to hemolysin E (HlyE; ClyA) from E. coli [13][14][15][16], which forms pores in target cell membranes by homooligomerization [17][18][19]. Nonetheless, all three Hbl components are necessary for maximum biological activity [3] and thus, to form the pore. According to the current model, the three components bind to the target cell surface sequentially in the binding order B-L1-L2 [9,12]. A defined concentration ratio is also required with most efficient pore formation at a ratio of Hbl L2:L1:B = 1:1:10 or 10:1:10 [12]. Next to the sequential binding, complex formation in solution between Hbl B monomers, Hbl B and L1, as well as Hbl L2 and L1, is also essential for rapid pore formation. It has been shown that complexation with Hbl L1 increases binding of Hbl B to the target cell surface [12,20,21]. The Hbl pore has recently been shown to be moderately cation selective and rather unstable [20]. The specific Hbl target structures LITAF and CDIP1 have also just recently been discovered on the cell surface [22].
The hbl operon encoding the toxin components is found in approximately 40-70% of all known enteropathogenic B. cereus strains [23][24][25][26][27][28]. There are two alternatives to the hbl operon. The wider spread one consists of the genes hblC (encoding Hbl L2), hblD (encoding Hbl L1), hblA (encoding Hbl B) and hblB (encoding Hbl B'). The rarer variant misses the hblB gene. Several B. cereus strains harbor both variants and thus, express homologous sets of Hbl proteins [23,[29][30][31]. In B. cereus strain F837/76 only the hblCDAB operon exists [24,32], which is shown in Figure 1. 601 bp upstream of hblC is the transcription start [33]. The PlcR binding box is also depicted [34]. Next to the global phospholipase C regulator, expression of the hbl operon is further regulated, probably in a concerted action, by CcpA, CodY, SinR, ResD and Fnr [24]. The genes hblC, D, A, and B have a size of 1320, 1221, 1128, and 1398 bp. Between hblC and D is a space of 61 nucleotides, between hblD and A 36, while the genes hblA and B are separated by 402 nucleotides. Upstream and downstream of hblB, a stem loop has been identified, which could function as transcriptional terminator [9]. Furthermore, a PlcR-independent transcription of hblB has been suggested [35]. hblB was first found by sequencing the hblA gene of B. cereus strain F837/76 [36]. As the C-terminal part of hblB and hblA differs significantly, it was stated that hblB emerged from duplication of hblA and a C-terminal fusion with another ORF [34].
Transcription of hblCDA was found to end within [33,37,38] or upstream [9] of hblB, until Clair et al. demonstrated that the gene is transcribed, translated, and the protein exported [35]. The study also suggested that Hbl B' accumulates in B. cereus supernatants in lower amounts than the other Hbl components, which might be a reason that it has not been detected earlier. Furthermore, a 2-fold upregulation of hblB in hyperflagellated swarm cells of B. cereus strain ATCC 14579 was found [39]. Although it has now been shown that Hbl B' is indeed produced and secreted by B. cereus, it is still unclear if it has a certain function. Due to the sequence homology of Hbl B, it has been speculated that Hbl B' could complement or reinforce the function of Hbl B under specific growth conditions [35].
Just recently, we were able to overexpress and purify fully functional recombinant Hbl components (rHbl) from E. coli and to study the mode of action of Hbl in more detail [12,20,21]. The aim of the current study was (i) to develop a system for the detection of Hbl B' in B. cereus culture supernatants and (ii) to investigate its putative involvement in Hbl activity and thus, in virulence of B. cereus.  23,[40][41][42][43]. For RNA preparation, the strains were cultivated for 3 h in CGY (casein-glucose-yeast) medium with 1% glucose before centrifugation (15 min, 4°C 4000 g) and disposal of the supernatant. For a collection of toxinrich supernatants, the strains were grown for 6 h in CGY medium. After centrifugation, 1 mM EDTA was added, and the supernatants were filtered through 0.2 mm sterile filters. For the overexpression of the rHbl components L2, L1, B and B,' the E. coli strain BL21 (DE3) transformed with the corresponding pASK-IBA plasmids was grown in LB medium with 100 µg/ml ampicillin.   [28,32]. hblC and D are separated by 61, hblD and A by 36 bp. Between hblA and B is a space of 402 bp. Upstream and downstream of hblB, a stem loop has been detected [9]. The promoter of the hblCDA operon is shown, including the transcription start TAA [33]. 236 bp upstream of the promoter, a binding box of the global regulator protein PlcR has been identified [34]. The figure was adapted from Dietrich et al [24].

Cell lines and culture conditions
Vero cells were purchased from ECACC (European Collection of Cell Cultures), CaCo-2 and A549 cells from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). They were maintained in 80 cm 2 culture flasks in a humidified incubator at 37°C and 7% CO 2 as recommended by the supplier.

Preparation of total RNA
Total RNA preparation and on-column DNase digestion were performed using peqGOLD Total RNA and peqGOLD DNase I Digest Kit (peqlab, Erlangen, Germany) according to the instructions of the manufacturer. Electrophoresis on agarose gels and testing in a spectrophotometer revealed the quality of the RNA. RNA purity was confirmed via PCR for a 241 bp 16S rRNA fragment using the primers 16S_fw (GGAGGAAGGTGGGGATGACG) and 16S_rev (ATGGTGTGACGGGCGGTGTG).

Reverse transcription and hblB PCR
Using 100 ng RNA as template, double-stranded cDNA was synthesized with the ProtoScript firststrand cDNA synthesis kit (New England Biolabs, Frankfurt, Germany) and random primer mix according to the instructions of the manufacturer. The cDNA was subsequently applied to a PCR for a 166 bp fragment of hblB using the primers hblB-3'-fw (CATAACG CATACACTTTTGAAATAAAG) and hblB-3'-rev (CCGCAAATTCATCATTTGGAT TG). The fragment represents the 3' part of the gene not identical to hblA.

Cloning, overexpression, and purification of recombinant Hbl B'
Recombinant Hbl L2, L1, and B were produced as described before  and HblB'-rev-NcoI (ATATCCATGGTCAGTTCAT TATATTTTGTACTTTG). The amplified gene lacked the sequence for the N-terminal signal peptide for secretion [34]. It was cloned into the vector pASK-IBA5plus (IBA Lifesciences, Göttingen, Germany), which added an N-terminal strep-tag to the recombinant Hbl B' protein. Analogously to that, a truncated rHbl B' protein was produced lacking the C-terminal 91 amino acids. For that, the same forward primer as well as HblB'(tr)-rev-NcoI (ATATCCATGGTCAAT TCAAAAAATCCTGTTCTTTTTTCTC) as reverse primer was used. Similarly to the full-length hblB of F837/76, the hblB genes of the other 10 B. cereus strains (see above) were amplified and cloned. Overexpression and purification of the rHbl B' proteins were performed analogously to Hbl L2, L1, and B as described before [21].

SDS PAGE and Sypro staining
SDS-PAGE was run on a PhastGel gradient (10-15%) minigel system (GE Healthcare, Munich, Germany). For Sypro staining, proteins were fixed on the gel for 2 × 30 min in 50% MeOH and 7% acetic acid. The gel was then incubated with 2 ml Sypro Ruby protein stain (Sigma-Aldrich; Merck, Darmstadt, Germany) overnight at room temperature. After 30 min washing in 10% MeOH and 7% acetic acid and additional washing for 10 min in H 2 O, fluorescence signals were detected on a UVP ChemStudio imager (Analytik Jena, Jena, Germany).

Western blotting
For Western blotting, 30 µl protein solution or B. cereus culture supernatant were applied to Criterion XT precast gels (BioRad Laboratories, Feldkirchen, Germany). After electrophoresis, proteins were blotted to a PVDF-P membrane (Millipore; Merck, Darmstadt, Germany). The membrane was saturated with 3% casein-PBS and incubated for 1 h at room temperature with 2 µg/ml Strep-MAB-Classic (IBA Lifesciences, Göttingen, Germany) or monoclonal antibody (mAb) 11A5 [21] for detection of Hbl B.' Afterward, it was washed three times in PBS with 0.1% Tween 20 and incubated overnight with a 1:2000 dilution of rabbit anti-mouse-horseradish peroxidase conjugate (Dako; Merck, Darmstadt, Germany) before three further washing steps in PBS with 0.1% Tween 20 and two in PBS. Subsequently, Super Signal Western Femto (Pierce; Thermo Fisher Scientific, Waltham, MA, USA) was applied, and chemiluminescence signals were sensed on a UVP ChemStudio imager (Analytik Jena, Jena, Germany).

Determination of hemolytic activity
Ten µl of B. cereus supernatants or rHbl components (1.5 pmol/µl each) were applied as mixtures or individually into 3.5 -mm diameter stamp holes on sheep blood agar plates. If not stated otherwise, they were photographed after 3-6 h of incubation at 32°C. For quantification, samples were applied to hemolysis assays with defibrinated sheep blood (Oxoid; Thermo Fisher Scientific, Waltham, MA, USA). The blood was applied to centrifugation (5 min at RT and 590 g) and washed three times in 150 mM NaCl, 5 mM Tris-HCl, pH 7.2. After that, it was diluted to 2% in the buffer. Two µl B. cereus supernatant or 10 µl rHbl samples were added to 800 µl total volume. As negative and positive controls, erythrocytes in 800 µl buffer and in 800 µl H 2 O were carried along, respectively. Samples (triplicates) were incubated for 20 min (supernatant) or 30 min (rHbl) at 37°C and subsequently centrifuged for 1 min at RT and 8000 g. Subsequently, an Infinite F50 photometer (Tecan Group Ltd., Männedorf, Switzerland) was used to determine the optical density at 562 nm. Results were compared to the positive control, which was set to 100%.

Flow cytometry
1 × 10 6 Vero cells in 500 µl buffer (140 mM NaCl, 15 mM HEPES, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM glucose, pH 7.2) per sample were used. rHbl B, rHbl B', and rHbl B'(tr) were applied at a concentration of 9.38 pmol/ml in the cell culture medium to a total volume of 1 ml and incubated for 1 h at 37°C under gentle agitation. Similarly, 1:1 mixtures of rHbl B and L1, L2, or B' were used. After two washing steps in 2 ml 1% BSA-PBS and centrifugation for 5 min at 800 rpm, 5 µg/ml mAb 11A5 was added for 1 h at RT. Samples were again washed twice and subsequently incubated with 2 µg/ml Alexa Fluor® 488 goat antimouse IgG (Life Technologies; Thermo Fisher Scientific, Waltham, MA, USA) for 45 min at 4°C. Alternatively, directly labeled mAbs 1B8-or 1G8-Alexa Fluor® 488 were used. Two additional washing steps were carried out. After that, cell pellets were resuspended in 500 µl 1% BSA-PBS and transferred to flow cytometry tubes. FACS Calibur and CellQuestPro software (BD Bioscience, Heidelberg, Germany) were used for fluorescence measurements. Results were analyzed using Flowing Software (Turku Bioscience, https://bioscience.fi/services/cell-imaging/flowingsoftware/). Cell populations were visualized in the FSC SSC (forward scatter -side scatter) dot plot. Fluorescence was recorded in FL1 (fluorescence channel 1) and is shown as histogram.

Surface Plasmon Resonance (SPR)
Measurements were performed on a Biacore S200 instrument (Cytiva Europe GmbH, Freiburg, Germany). rHbl B' was diluted to a final concentration of 150 nM in 10 mM sodium acetate, pH 4.8, and chemically immobilized (amine coupling, 350 RU bound) onto series S sensor chip CM5 (Cytiva Europe GmbH, Freiburg, Germany). rHbl L1, L2, and B protein samples were diluted in a running buffer (PBS, 1 mM DTT and 0.005% Tween 20) to the final concentration of 7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 µM, and 2 µM and injected over the sensor chip surface at 30 µl/min at 20°C from the lowest to the highest concentration. Injection of 250 nM protein concentration was always performed in duplicate within each experiment. For the sensor chip regeneration after each injection, 1 M MgCl 2 solution has been used for rHbl L1 and 0.5 M NaCl solution for rHbl L2 and B. In order to subtract any background noise from each experiment, all samples were also run over an unmodified sensor chip surface. Data analysis was performed with Biacore S200 evaluation software v1.1. For each measurement, the equilibrium dissociation constant (K D ) was calculated using a 1:1 binding model. The K D s from three experiments were used to calculate the mean values of these variables and the SEM.

In silico analyses
DNA and protein sequence alignments were generated using clustal omega [44]. Structural models of Hbl B and B' of B. cereus strain F837/76 were gained using SWISS-MODEL [45].

The hblB gene is expressed in 12 different B. cereus strains
Earlier, contradictory studies [28,35] raised the question if the hblB gene is generally transcribed and the corresponding protein expressed in B. cereus. To answer that, total RNA was prepared from different B. cereus strains with known enterotoxin gene profiles [23,40,42], which were grown for 3 h in CGY medium (Figure 2a). After a control PCR for a 16S rRNA gene fragment revealed no residual DNA (Figure 2b), RNA was reverse transcribed into cDNA and subsequently used for PCR amplification of a 166 bp fragment of hblB. The hblB fragment could be detected for all tested B. cereus strains (Figure 2c), indicating that hblB is indeed transcribed under ideal growth conditions. Moreover, the hblB fragment was also detected when cDNA from a previous study was used, in which strain F837/76 was grown under "simulated intestinal conditions" (RPMI 1640 medium, 37°C 7% CO 2 atmosphere) [43]. As a positive control, chromosomal DNA of strains F837/76 and INRA A3 was used. Chromosomal DNA of Nhe reference strain NVH 0075-95 [48] as well as MHI 226 bearing only a truncated hblCDA operon [23] revealed the specificity of the reaction (see also Figure 2c).

mAb 11A5 detects recombinant (r)Hbl B and rHbl B' in Western blots
Analogously to the three recently purified rHbl components [21], the hblB gene from B. cereus strain F837/76 was cloned into the vector pASK-IBA5plus. Thereby, the N-terminal signal peptide for secretion was dismissed, and an N-terminal strep-tag was added. E. coli BL21 (DE3) was used for protein overexpression. Purification was conducted via affinity chromatography. The 460 amino acid protein with a predicted molecular mass of 51.5 kDa was detected by Western blotting using an antistrep mAb (Figure 3a). Subsequently, the generated rHbl B' protein was used to screen a set of Hbl B-specific mAbs [21,46] for binding. Of the three potential candidates, mAb 11A5 was best suited for the specific detection of rHbl B and B' in Western blots at 40.9 and 51.5 kDa, respectively. From the band intensities, the relative affinity of mAb 11A5 was estimated, which was approximately 3x higher for rHbl B than B' (Figure 3).  Western blots were performed using specific mAb 11A5. After 20 s exposure, strong signals for Hbl B appeared. Thus, the membrane was cut and Hbl B'-specific signals were detected after an additional 3 min exposure. This procedure is possible due to the molecular weights of Hbl B (approx. 40 kDa) and Hbl B' (approx. 50 kDa). The recombinant proteins, which were used as concentration standards, appeared slightly bigger in the gel than the native proteins, which is due to their additional strep-tag (refer to Figure 3).  L2, L1, and B have already been shown in PI (propidium iodide) influx tests and WST-1 (water-soluble tetrazolium) bioassays on target cells, as well as on planar lipid bilayers [12,20,21]. Because of the sequence homology between Hbl B and Hbl B' [34] , the latter was considered as a putative substitute for at least Hbl B. In Figure 5 it is shown that rHbl B' is neither able to replace rHbl B nor rHbl L2 or L1.
This observation was made in WST-1 bioassays ( Figure 5), where cell viability is determined after 24 h of incubation, as well as in PI influx tests ( Figure 5), where pore formation into the target cell membrane is measured. Consistent data were obtained on Vero, CaCo-2, and A549 cells (see Figure 5), indicating that the failure of rHbl B' to substitute the other components does not depend on the target cell line. Also, no hemolytic activity was observed on sheep blood agar plates when rHbl B was substituted with rHbl B' ( Figure 5).

rHbl B' reduces cytotoxic and hemolytic activity of Hbl
After showing that rHbl B' cannot substitute for any of the other rHbl proteins, all four Hbl components were applied simultaneously. Increasing ratios of rHbl B' from 1x to 10x compared to the other three components led to decreased titers in WST-1 bioassays on Vero cells (Figure 6), suggesting that the presence of rHbl B' reduces cytotoxic activity of Hbl. Similar observations were made in PI influx tests. Increasing ratios of rHbl B' of up to 10x significantly delayed PI influx into Vero cells caused by rHbl L2+L1+B ( Figure 6) and also by the supernatant of B. cereus strain F837/76 ∆nheABC ( Figure  6). When BSA was used instead of rHbl B' (see Figure 6), no delay in PI influx was determined, indicating that the inhibitory effect is highly specific to rHbl B.' An inhibitory effect of rHbl B' was also seen on CaCo-2 and A549 cells ( Figure 6). It is noteworthy that strain F837/76 (as well as its nhe deletion variant) was initially used for the experiments and especially for cloning of hblB, as it is well sequenced and annotated. Nevertheless, in the course of this study, it was shown that in this particular strain, the hblB gene is indeed transcribed, but the corresponding Hbl B' protein is not expressed in detectable levels in the supernatant (see above). rHbl B' also showed an influence on hemolytic activity of Hbl on sheep blood agar. When a mixture of rHbl L2, L1, and B was applied together with rising amounts of rHbl B,' the continuous hemolysis around the stamp hole disappeared. In a quantitative assay with defibrinated sheep blood, rHbl L2+L1+B caused approx. 20% hemolysis compared to the positive control, which was diminished under addition of rising amounts or rHbl B' (Figure 7).  The same observations were made for supernatants of strains F837/76 and F837/76 ∆nheABC. While the Hblprovoked outer hemolysis circle on sheep blood agar disappeared with rising amounts of rHbl B,' total hemolysis decreased from approx. 50% to 10% (Figure 7). When rHbl B' was applied into a separate stamp hole with 2-3 mm distance to the supernatant of F837/76, the outer hemolysis circle seemed to be pushed inwards ( Figure  7). This was compared to the previously detected effects of the other three rHbl components [12] on supernatant of strain F837/76 ∆nheABC. While rHbl L2 had no effect on the outer circle, rHbl L1 erased and rHbl B enlarged it. The circle was again pushed inwards by rHbl B.'

rHbl B' inhibits interaction of rHbl L1 and B instead of B binding to the cell surface
To gain evidence at which step of Hbl complex/pore formation rHbl B' might interfere, the rHbl components were applied consecutively in cytotoxicity experiments (seeFigure 8). First, Vero cells were incubated for 2 h with rHbl B' and then washed in cell culture medium before application of a dilution series of rHbl L2+L1+B for further 4 h and addition of WST-1. The resulting reciprocal titers showed a slight, but non-significant reduction of cytotoxic activity ( Figure  8), giving the initial idea that rHbl B' does not bind to  Figure S1. Here, one representative overlay histogram is depicted, respectively. the Vero cells and does not inhibit binding of rHbl B to the cell surface. This was supported by PI influx tests, in which the rHbl components were applied in 1:40 dilution individually and consecutively to the Vero cells. No significant differences were seen when rHbl B' was not present or applied first or after rHbl L1. On the other hand, a clear delay of PI influx appeared when rHbl B' was added after B and before L1 (Figure 8). These data further pointed to the conclusion that rHbl B' influences the interaction of rHbl B and L1 rather than the interaction with L2 or binding of B to the target cell surface. Flow cytometric analyses supported the assumption that rHbl B'despite its sequence homology to B -is not able to bind to the target cell surface. Only 4% of Vero cells treated with rHbl B' were fluorescence-positive, while approximately 30% of cells treated with rHbl B showed fluorescence (Figure 8). In a second approach, mAb 1B8 was used for detection, which is specific to Hbl B, but none of the other Hbl proteins [46]. Thirty-minute incubation with the rHbl proteins resulted in 54% fluorescence-positive cells for rHbl B, and 51% for rHbl B+L2. As observed before [12], addition of rHbl L1 enhanced the binding of B to the cell surface (95%). A slight increase in fluorescencepositive cells was also observed with rHbl B+B' (64%) (Figure 8). For that reason, a third experiment was conducted using Hbl B-specific mAb 1G8 for detection, which is cross-reactive with L1, but not with B' [21]. Here, no significant differences were detected between rHbl B and rHbl B+B' (Figure 8). These experiments allow us to conclude that it is unlikely that rHbl B' binds to the target cell surface or that it interferes with cell binding of B.

rHbl B' directly interacts with rHbl L1
According to previous results, the interaction of Hbl B and L1 is an important prerequisite for rapid pore formation [12,21]. The above shown data suggested that Hbl B' hinders this interaction either by binding to Hbl B or L1. Application of rHbl L1-B' mixtures to enzyme immunoassays and native blotting indicated first evidence for complex formation between Hbl L1 and B' (data not shown). Surface plasmon resonance (SPR) measurements performed similarly to earlier studies [21] proved this initial observation and verified the binding of rHbl B' to rHbl L1. Coupling of rHbl B' to the sensor chip and addition of increasing concentrations of rHbl L1 resulted in a K D value of 130.6 ± 47.6 nM. For rHbl L2 and B, little, rather unspecific interaction was observed, from which K D values could not be calculated (Figure 9).

Truncated rHbl B' protein does not exhibit Hbl B function
After it became clear that Hbl B' indeed interferes with pore formation and cytotoxicity of Hbl, the question arose whether this is due to its extended C-terminus presumably developed from duplication  [46] of hblA and the 273 bp longer hblB gene. B. Amino acid sequence alignment using clustal omega [46] of the corresponding Hbl B and Hbl B' proteins. C. Structural comparison. Protein models were gained using SWISS-MODEL [47]. The Nterminus is in the upper part of each image, the C-terminus is centred in the front of each image, and the C-terminal amino acid is highlighted. Hbl B: Sequence identity to Hbl B with solved crystal structure (PDB ID: 2nrj; [15]) = 99.42 %. Hbl B': 2nrj was also used as template, sequence identity = 71.39 %. For the C-terminal part, which is not similar to Hbl B (refer to A and B), no template exists and thus, it could not be modeled.
of hblA and a C-terminal fusion with another ORF [34]. Figure 10a shows an alignment of the 1128 bp hblA gene and the 273 bp longer hblB gene from strain F837/76. In Figure 10b, the corresponding amino acid alignment is depicted. An analysis of the corresponding protein structures revealed high structural similarities between Hbl B and Hbl B.' It is noteworthy that the C-terminal part, which does not match Hbl B, could not be modeled, as no template exists (Figure 10c).
To test if that part of Hbl B,' which equals Hbl B, exhibits a similar function, a truncated version (tr) of rHbl B' was produced lacking the C-terminal 91 amino acids. Cloning, protein overexpression and purification were performed analogously to the fulllength rHbl B' (Figure 11a). Similarly, rHbl B'(tr) was applied to a selection of the tests described above. Like the full-length rHbl B,' the truncated version was not able to replace any of the other protein components in the Hbl complex, as shown by PI influx and WST-1 assays and in hemolysis tests on blood agar plates (Figure 11b). Moreover, the protein did not bind to Vero cells (Figure 11c). Again comparably to the full-length protein, the  Figure 5), as they were obtained in a later approach with less active PI. (c). Flow cytometric analysis of rHbl B'(tr) on Vero cells using mAb 11A5. Black: only Vero cells. Dark gray: negative control without antigen. Light green: rHbl B as control. Fluorescence intensities vary compared to a similar approach (see Figure 8c), as protein incubation times were extended. Blue: rHbl B' as control. Cyan: rHbl B'(tr). One representative overlay histogram as well as means and standard deviations (fluorescence-positive cells) of triplicates are shown. Additional flow cytometry data can be found in Figure S1. (d). Addition ofrising amounts of rHbl B'(tr) to rHbl or supernatant of strain F837/76 on blood agar plates. Plates with the rHbl samples were photographed after 24 h incubation at 32°C. truncated version hindered the hemolytic activity of rHbl or the supernatant of strain F837/76 ( Figure  11d). Altogether, despite the similar structure, Hbl B' seems not to be able to execute Hbl B function, not even when truncated to the same size. This allows us to conclude that not only the additional 91 amino acids C-terminal part, but especially amino acid variations in the N-terminal part of the protein (refer to Figure 10) account for the functional difference.

Purified Hbl B' proteins of nine strains repress Hbl activity with varying strength
Following the fact that Hbl B' is produced and secreted in nearly all of the tested B. cereus strains, we were interested to see if these proteins exhibit comparable or varying inhibitory activities toward Hbl toxicity. As the hblB genes and thus, the Hbl B' proteins of strains 6  c), the Hbl B' proteins of eight further strains were recombinantly expressed and purified analogously to F837/76 (see above). Figure 12 shows the Hbl B' proteins stained by Sypro Ruby and detected via StrepMAB-Classic and mAb 11A5 in Western blots. The rHbl B' proteins of all strains were detected at their predicted size, except strain F3175/03 (D7), for which -despite no stop codon in the DNA sequence -a truncated protein of approx. 37 kDa appeared in all tests (Figure 12a). When the purified rHbl B' proteins were applied in stamp holes on sheep blood agar plates analogously to rHbl B' of strain F837/76 (compare Figure 7), they inhibited hemolysis of F837/76 ∆nheABC supernatant until a dilution of 1:4, as well as hemolysis of rHbl L2+L1+B (data not shown). Strainspecific differences were first observed when 10 µl rHbl B' were pre-incubated in the stamp holes for 1 h before application of 10 µl rHbl L2+L1+B. rHbl B' of strains INRA A3 and RIVM BC 934 strongly inhibited hemolysis, while only a weak effect was observed for rHbl B' from strain 14294-3 (M6). Inhibition of hemolysis with rHbl B' of all other strains was interjacent (see Figure 12b). These strain-specific differences in rHbl B' impact on Hbl pore formation were further investigated in PI influx tests. Generally, PI influx and thus, pore formation was delayed with increasing amounts of rHbl B' (1x, 5x, and 10x surplus compared to rHbl L2+L1+B or supernatant of strain F837/76 ∆nheABC were tested; data not shown). Differences between the rHbl B' proteins of the nine strains were most obvious at rHbl B:'F837/76 ∆nheABC = 1:1 as well as 5x surplus of rHbl B' compared to rHbl L2+L1+B (see Figure 12c (Figure 12d). For a better overview and comparison of the data, these results are summarized in Table 1.
Altogether, our data suggest that the fourth component of the hbl operon, Hbl B,' indeed influences Hbl complex and pore formation. Concurrently, significant strain-specific differences occur underlying a yet not understood mechanism. Ongoing research points to different binding capacities toward Hbl L1, which is a very interesting subject for further investigations.

Discussion
Many years of research have led to a better understanding of the mode of action, especially the mechanism of pore formation, of the enterotoxin complex Hbl from B. cereus. It is clear that the three components Hbl B, L1, and L2 bind to the target cell surface in a defined order [9,12]. A defined concentration ratio as well as complex formation between the three single components prior to cell binding also determines the velocity of pore formation and cytotoxic activity [12,20,21]. But can a fourth protein component play an additional role in the mechanism of pore formation? Nearly all bacterial pore-forming toxins are composed of a maximum of two different components [49,50]. Until recently, the B. cereus enterotoxins Nhe (non-hemolytic enterotoxin) and Hbl were the only known pore forming toxins consisting of three individual protein components [38,51]. Then, orthologues were detected in Gramnegative bacteria, such as Serratia marcescens, Erwinia mallotivora, and Aeromonas hydrophila after extensive in silico search, and the mechanism of the tripartite αpore forming toxin AhlABC of A. hydrophila was characterized [52].
The fourth protein component, Hbl B,' seems not to be necessary for pore formation and cytotoxic activity of Hbl. This is justified by the occurrence of two distinct hbl operons among different strains or also within a single strain, the more common form hblCDAB and the rarer variant hblCDA missing Hbl Table 1. Effects of rHbl B' proteins from nine B. cereus strains on Hbl activity. Summary of hemolysis and pore formation experiments depicted in Figure 12. ++: strong inhibitory effect, +: inhibitory effect, (+): weak inhibitory effect.. rHbl B' from strain Inhibition of hemolysis ( Figure  11) Inhibition of F837/76 ∆nheABC ( Figure 11) Inhibition of rHbl ( Figure  11 B' [23,[29][30][31]. Moreover, several studies, including our own, proved pore formation and cytotoxicity using the three recombinantly expressed proteins Hbl L2, L1, and B [9,12,20,21].  [23,40,42]. With this tool, Hbl B' could also be detected in the supernatant of 10 B. cereus strains, but with varying concentrations. Interestingly, strain F837/76 seemed not to express Hbl B' in a detectable amount, although hblB mRNA existed. In general, concentrations of Hbl B and presumably Hbl B' in the B. cereus supernatants obtained in Western blots in this study are higher than concentrations determined earlier using EIAs and purified proteins as concentration standards. In the latter one, average Hbl B concentrations of approx. .5 ng/µl were obtained (unpublished data). Just recently, we gained evidence that the concentration of Hbl B in B. cereus culture supernatants might be drastically underestimated when tested with EIAs, as the protein is highly complex, with itself as well as with Hbl L1 and L2 [20,21].
Furthermore, the present study showed, for the first time, an additional regulatory element in Hbl complex and pore formation via Hbl B'. It is the first one to gain Hbl B' as single, recombinantly expressed and purified protein. Hbl activity (cytotoxic or hemolytic) was completely abolished when either rHbl B or L2 or L1 was replaced with rHbl B'. Thus, hypotheses were disproved that Hbl B' might exhibit a similar function than Hbl B [35] due to their sequence homology [34] (see also Figure 10). Instead of sustaining toxicity, our data suggest that Hbl B' reduces Hbl activity. This was detected in PI influx tests, a method measuring pore-forming activity, as well as in WST-1 bioassays, which show the rate of dead target cells. Hemolytic activity of both rHbl and B. cereus culture supernatants was also minimized. To our knowledge, this is the first time that a distinct activity of the hblB gene product was observed. Furthermore, we showed that rHbl B' cannot bind to Vero cells and thus, does not compete with Hbl B for binding sites at the target cell surface. Moreover, it does not seem to influence the binding of B to the cell surface. On the contrary, it rather inhibits the interaction between Hbl B and L1. SPR measurements showed binding of rHbl L1 to rHbl B' with a K D value of 131 nM (see Figure 9), which is even stronger than binding of rHbl B to L1 observed earlier (K D value of 473 nM [21]), but still relatively low compared to the binding capacity of NheB-C (K D value of 0.48 nM [53]). Thus, the binding of Hbl B' and L1 seems to be strong enough to affect the interaction between Hbl B and L1.
It has been suggested that hblB has originated by duplication of hblA and a C-terminal fusion with another ORF [34]. Interactions of Hbl B' with Hbl L1 might occur because of its sequence homology to Hbl B. The present study further showed that the additional C-terminal 91 amino acids are not (exclusively) responsible for the protein's inhibitory function (compare Figure 11). We believe that this is rather based on amino acid sequence variations compared to Hbl B (see Figure 10). It has further been suggested that hblB has originated last in the evolution of the enterotoxins [23,34] and it has been kept, so it is most likely that an evolutionary advantage exists. According to the data obtained in the present study, it seems to be highly important to instate the Hbl pores precisely via an additional "control mechanism" by Hbl B'. In earlier studies, we showed that Hbl B is present in culture supernatants mostly in large complexes, either with L1 or self-complexed [20]. On the other hand, also a sufficient amount of free Hbl B is necessary for optimal pore formation [12,20], similarly as it has been shown for NheB [54]. Although complexation with Hbl L1 improves binding of Hbl B to the target cell surface, excess of L1 inhibits Hbl activity [12]. The role of the blocking interaction between Hbl B' and Hbl L1 might thus be to stabilize L1 to partly avoid the formation of huge complexes and to ensure a balance between B-L1 complexes and the free subunits.
In the present study, it was further shown that rHbl B' proteins from nine B. cereus strains decreased the hemolytic and pore-forming activity of Hbl. Only a truncated version of rHbl B' of strain F3175/03 (D7) could be recombinantly expressed and purified. Since it was detected by StrepMAB-Classic and mAb 11A5 in Western blots (see 12a), it can be concluded that the C-terminal part of the protein is missing. Similarly to, but less actively than the truncated protein rHbl B'(tr), it inhibited pore formation and hemolysis caused by Hbl (12b-d). This allows us to make the assumption that the N-terminal part of Hbl B,' which is rather similar to Hbl B (refer to Figure 10), is responsible for the interference of B' with Hbl activity by interacting with Hbl L1. A comparison of the Hbl L1 protein sequences of the different strains is depicted in Figure  S2a, which is highly conserved (compare also [42]). On the contrary, Hbl B' is far less conserved (compare Figure S2b and c). Thus, the genetic basis for the differences in the inhibition of Hbl activity is rather suspected here. Secondly, the high variety of Hbl B' production might also be found in genetic variations within the intergenic region between hblA and hblB (compare Figure S2d). Initially, it has been suggested that hblB is transcribed together with hblCDA as a polycistronic mRNA [28]. Recent studies have found a stem loop resulting in a putative transcriptional stop site upstream of hblB from B. cereus ATCC 14579 [35] and ATCC 10876 [9]. Clair et al. further identified putative −10 and −35 regions and thus suggested that hblB might be transcribed as a single unit [35]. Both of the putative stem loop sequences were identified in the upstream region of hblB of our 11 strains. The predicted −10 and −35 regions as well as the predicted transcription start site were identified for nine strains. In general, the length of the hblB upstream regions varies and is less conserved than the hblB genes ( Figure S2d). At this point, it can only be concluded that strain-specific variations in Hbl B' production and in its inhibitory activity exist, as well as in the sequences of hblB and especially the hblA-hblB intergenic region. Further in-depth analyses are necessary in order to connect those variations.
Nevertheless, this study is the first one to present a simple tool for the detection of the fourth component of the hbl operon, Hbl B,' in B. cereus culture supernatants. We found the protein to be secreted by 10 different strains. More importantly, we could verify an inhibitory function of Hbl B' in the mechanism of Hbl, which is most likely the balanced orchestration of free Hbl B and Hbl B-L1 complexes in solution.