Frontiers in Microbiology

In present study, we describe "tat-fimbriae (tafi)" - a novel type of archaeal surface appendages isolated from haloarchaeon Haloarcula hispanica. These filamental structures are unique because they are formed of protein subunits secreted through the twin-arginine translocation pathway (Tat-pathway), in contrast to well-known archaeal surface filamentous structures secreted by the general secretory pathway (Sec-pathway). No cases of the role of Tat-pathway in the assembly of archaeal and bacterial filamentous structures have been described to date. "Tafi" are the first example of such structures. The precursor of the major tafi protein subunit TafA contains the N-terminal signal peptide carrying a twin-arginine consensus motif and fimbria-forming mature TafA lacks this signal peptide. We analyzed the gene neighborhood of the tafA homologues in the known haloarchaeal genomes and found a conservative cluster of seven associated genes tafA, B, C, D, E, F, G. We assume that all of them take part in the tafi synthesis. TafC and TafE proteins, whose precursor sequences also contain twin-arginine motifs, were detected as minor components of tafi. TafE protein is structurally similar to TafA, while TafC contains a TafA-like N-terminal domain and a C-terminal "laminin G-like" domain capable of functioning as an adhesin. TafD is annotated as a signal peptidase I. The functions of TafB, TafF and TafG are not known yet. This study demonstrated that {Delta}tafA and {Delta}tafD deletion mutant strains synthesized archaella and not tafi, and only tafi were detected in {Delta}arlK (gene of common archaellin/pilin signal peptidase) deletion strain. It was shown that the expression of complete Har. hispanica taf-gene cluster in a heterologous host Haloferax volcanii that does not have similar genes leads to synthesis of recombinant tafi structures similar to the native ones. The tafi function remains elusive, but our preliminary data suggest that these structures may be involved in cell adhesion to different surfaces or substrates.

Inosine could potentially become a novel antibacterial agent against A. acidoterrestris as low-dose of inosine can prevent A. acidoterrestris contamination. However, until now the antibacterial mechanism of inosine targeting A. acidoterrestris is still unknown. In this study, to unravel the mechanism of inosine against A. acidoterrestris puzzle, the effects of inosine on bacterial surface hydrophobicity, intracellular protein content, cell membrane damage extent and permeability of the A. acidoterrestris were investigated. The results showed that inosine can effectively inhibit the growth and reproduction of A. acidoterrestris by destroying the integrity of cell membrane and increasing its permeability, causing the leakage of intracellular nutrients. Furthermore, the interaction networks of inosine target proteins were analyzed. The interaction networks further revealed that damage of bacterial cell membrane might be relevant to inosines effect on bacterial DNA replication and cell energy metabolism through regulating nucleotide synthesis and metabolism and the activity of translation initiation factors. Finally, the antibacterial mechanism of inosine against A. acidoterrestris was proposed.

Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa) is one of the most devastating diseases of kiwifruit and can damage almost all kiwifruit varieties. The severity of the disease occurrence is closely related to the temperature. Our previous research indicated that Psa showed stronger pathogenicity and expansion ability at relatively cool temperatures, but how Psa senses environmental temperature and regulates its virulence mechanism remains unclear. In this study, 69 Histidine kinases (HK) in Psa were predicted through bioinformatics analysis, and 9 differentially expressed HK genes were identified at varying temperatures through pathogenicity detection and quantitative reverse transcription PCR (qRT-PCR). Among them, HktS as a temperature signal receiver was identified, and its response regulator (RR) HktR was determined through structure analysis and cotranscription assay. The results showed that HktR can bind to transcription factor RpoD, and RpoD bind to hrpRS promoter region, thus initiating the expression level of the type III secretion system (T3SS), which plays an important role in the pathogenesis of Psa. In addition, the motility of Psa was also regulated by HktS-HktR in a temperature-dependent manner. These findings reveal the molecular mechanism by which HktS-HktR acts as a temperature sensor to regulate bacterial virulence and motility of Psa, providing a new potential target for KBC control.

The family Natrialbaceae is a member of the class Halobacteria of the archaeal phylum Euryarchaeota. Seventeen genera with validly or effectively published names are currently included within this family. In this study, using pairwise average nucleotide identity and average amino acid identity comparisons in conjunction with phylogenetic analysis, it has been shown that the family Natrialbaceae is highly diverse and contains several potentially novel species and genera that are yet to be fully characterized. The deduced proteome sequence-based phylogenetic tree, constructed using the alignment- and parameter-free method CVTree3, contained six major clades, with Salinarchaeum sp. Harcht-Bsk1 being the only representative within clade 1. Furthermore, Haloterrigena daqingensis was found to be closely related to Natronorubrum sediminis, and it is proposed that these archaea together represent a novel genus. Interestingly, Haloterrigena jeotgali, Haloterrigena thermotolerans, and Natrinema pellirubrum were found to be very closely related to each other, and it is proposed that they be merged into a single species. Notably, the type genus Natrialba itself appeared to be heterogenous and contains species that could be broadly classified among two genera. Likewise, the genus Natrinema is also heterogenous and contains species that could be classified among six genera. Altogether, 19 novel genera have been proposed to be created, and four haloalkaliphilic archaea hitherto recognized only using genus names are confirmed to represent novel species.

Acid-responsive proteome expression profiles of Alicyclobacillus acidoterrestris (A. acidoterrestris) were analysed using label-free quantitative mass spectrometry to investigate its acid resistance mechanism at sublethal pH. Totally, 325 differential expression proteins were identified during acid stress at pH2.5 condition for 15 min, of which the expressions of 83 proteins were up-regulated and the other 242 proteins expressions were down-regulated. Differentially expressed proteins were mainly involved in organic nitrogen compounds metabolism, small molecule metabolism, organic acid metabolism and signal transduction. Overall, they were mapped into 97 metabolic pathways. Combination of KEGG pathway analysis and protein functional analysis suggested that the pH homeostasis system, changes in metabolic pathways, cell membrane permeability and DNA repair are the main acid resistance mechanisms of A. acidoterrestris at sublethal pH conditions. It is speculated that A. acidoterrestris may sense and transmit pH signals from the external environment through the nhaB protein which holds histidine-dependent acid resistance system, initiating a series of acid tolerance reactions. Our study demonstrated global physiological response of A. acidoterrestris to sublethal pH, which provided a better understanding of acid adaption mechanism of A. acidoterrestris.

Fusobacterium nucleatum (F. nucleatum, Fn) has emerged as a significant bacterium linked to cancer. However, the precise mechanisms by which Fn drives the progression of oral squamous cell carcinoma (OSCC) remain obscure. Outer membrane vesicles (OMVs), small vesicular structures secreted by Gram-negative bacteria, are known to contribute to their pathogenicity. This study investigated the role of Fn OMVs in oral cancer progression and the underlying mechanisms. MethodsMetagenomic sequencing was conducted on both oral cancer and control tissues. OMVs were isolated through ultracentrifugation. Cell proliferation assays on two cancer cell lines assessed the impact of Fn and its OMVs. Fn OMVs were administered to mice to evaluate their influence on tumorigenesis. RNA-seq was performed on OMV-treated cells to identify enriched genes, which were validated via qPCR and Western blot. Knockdown experiments were executed in vitro and in vivo to confirm the involvement of Fn OMVs in TNFR1-mediated NF-{kappa}B signaling. ResultsFn prevalence was markedly higher in oral cancer tissues compared to non-cancerous samples. In vitro, Fn and its OMVs significantly enhanced the growth and proliferation of oral cancer cell lines. Mice treated with Fn OMVs developed larger tumors than those receiving Fn alone. Furthermore, Fn OMVs notably induced TNFR1 expression and activated the NF-{kappa}B signaling pathway, as confirmed by RNA-seq, qPCR, and Western blot. TNFR1 knockdown significantly diminished the effects of Fn OMVs on cancer cell proliferation in vitro and tumor formation in vivo. ConclusionThis study compellingly demonstrates that Fn OMVs are pivotal in OSCC progression by directly activating TNFR1-mediated NF-{kappa}B signaling. These findings pave the way for future research into the prevention and treatment of oral cancer.

Subclinical mastitis is one of the most widespread diseases affecting dairy herds with detrimental effects on animal health as well as on milk productivity and quality. Despite the multi-factorial nature of this intramammary infection, the presence of pathogenic bacteria is regarded one of the main drivers of subclinical mastitis, leading to a disruption of the homeostasis of the bovine milk microbial community. However, the bovine milk microbiota alterations associated with subclinical mastitis still represents a largely unexplored research area. In this context, the species-level milk microbiota of a total of 75 milk samples, collected from both healthy and subclinical mastitis-affected cows from two different stables, was deeply profiled through an ITS, rather than a traditional, and less informative, 16S rRNA gene microbial profiling-based sequencing. Surprisingly, the obtained data of the present pilot study, not only revealed that subclinical mastitis is characterized by a reduced number of species in the bovine milk microbiota, but also that this disease does not induce standard alterations of the milk microbial community across stables. In addition, a flow cytometry-based total bacterial cell enumeration highlighted that subclinical mastitis is accompanied by a significant increment in the number of milk microbial cells. Furthermore, the combination of the metagenomic approach and total bacterial cell enumeration allowed to identify different potential microbial marker strictly correlated with subclinical mastitis across stables.

Daily cotrimoxazole (TMP/SXT) prophylaxis is part of the HIV treatment package for all new HIV-infected individuals in Uganda. Although this treatment has shown reduced morbidity and mortality in HIV, it remains controversial due to its contribution to developing antibiotic-resistant bacteria. Moreover, the effects of daily use of a broad-spectrum antibiotic on the gut microbiome remain unknown. To study the early effects, we analysed shotgun metagenome sequence data from stool samples of five newly HIV-infected individuals initiating TMP/SXT prophylaxis longitudinally for the first 30 days of treatment. Using shotgun metagenomics sequencing, we generated both taxonomic and functional profiles from each patient and compared gut microbial changes Pre-TMP/SXT and post-TMP/SXT on Day 5, Day 14, and Day 30. Daily TMP/SXT prophylaxis resulted in a shift characterised by an enrichment of Prevetollea and Ruminococcus genera members and the depletion of Lactococcus and Bacteroides genera members. Furthermore, these microbial shifts were associated with changes in the functional profile revealed by a differential abundance of pathways of amino acid metabolism, carbohydrate metabolism, and nucleotide biosynthesis linked to members of the Bacteroidaceae and Enterobacteriaceae families. TMP/SXT daily prophylaxis in HIV-infected individuals is associated with dramatic changes in microbial composition and functional profiles; however, other factors such as Age, Gender, HIV clinical stage, and ART regiment are at play. Further investigation is needed to examine the implication of these shifts on clinical management and outcomes among HIV patients.

Bacterial vaginosis (BV) is a common infection of the lower genital tract with a vaginal microbiome dysbiosis caused by decreasing of lactobacilli. Previous studies suggested that supplementation with live Lactobacillus may benefit the recovery of BV, while the outcomes vary in people from different regions. Herein, we aim to evaluate the effectiveness of oral Chinese-origin Lactobacillus with adjuvant metronidazole (MET) on treating Chinese BV patients. In total, 67 Chinese women with BV were enrolled in this parallel controlled trial and randomly assigned to two study groups: a control group treated with MET vaginal suppositories for 7 days and a probiotic group treated with oral Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 as an adjuvant to MET for 30 days. By comparing the participants with Nugent scores [&ge;] 7 and < 7 on days 14, 30, and 90, we found that oral administration of probiotics did not improve BV cure rates (57.14% and 67.74% at day 14, 57.14% and 58.06% at day 30, 32.14% and 48.39% at day 90 for probiotic and control group respectively). However, the probiotics were effective in restoring vaginal health after cure by showing higher proportion of participants with Nugent scores < 4 in the probiotic group compared to the control group (87.50% and 71.43% on day 14, 93.75% and 88.89% on day 30, and 77.78% and 66.67% on day 90). The relative abundance of the probiotic strains was significantly increased in the gut microbiome of the probiotic group compared to the control group at day 14, but no significance was detected after 30 and 90 days. Also, the probiotics were not detected in vaginal microbiome, suggesting that L. gasseri TM13 and L. crispatus LG55 mainly acted through the gut. A higher abundance of Prevotella timonensis at baseline was significantly associated with long-term cure failure of BV and greatly contributed to the enrichment of the lipid IVA synthesis pathway, which could aggravate inflammation response. To sum up, L. gasseri TM13 and L. crispatus LG55 can restore the vaginal health of patients recovering from BV, and individualized intervention mode should be developed to improve BV cure rates.

Bacillus cereus is a Gram-positive bacterium widely distributed in food, soil, and plants. It is a spore-forming, facultative anaerobic bacterium known as a food-borne opportunistic human pathogen responsible for causing gastrointestinal and non-gastrointestinal infections, including wound-associated infections. In the present study, we report that among single-component, bi-component, and tripartite alpha pore-forming toxin (-PFT) producing bacteria, the tripartite NheABC toxin produced by B. cereus induced the maximum cell death in infected epithelial cells. Similar to its effects in 2D monolayers, NheABC exhibited potent cell toxicity in 3D spheroids and intestinal organoids, targeting their cell membrane and mitochondria. Moreover, using erythrocytes as a model system, we found that the cytolytic activity of NheABC is pH-dependent, and was markedly reduced at acidic pH (5.5). The pH-dependent biological activity of NheABC was further confirmed by a liposome leakage assay. Importantly, NheABC enhanced the colonization of B. cereus in a non-gastrointestinal murine wound infection model. Overall, our study highlights the role of pH in regulating NheABC-mediated cytotoxicity in mammalian cells, which may lead to the development of novel therapeutic strategies for managing B. cereus gastrointestinal and non-gastrointestinal infections.

Staphylococcus aureus is a notorious pathogen responsible for significant morbidity and mortality in both human society and animal husbandry. The presence of S. aureus persisters is also one of the leading causes of recurrent and chronic diseases. Persisters are a subset of growth-arrested bacteria within a susceptible bacterial population that are able to tolerate antibiotic treatment and resuscitate after stress removal. Consequently, investigating their formation and characteristics is of crucial importance to provide mechanism-based options for their eradication. However, one challenge in mechanistic research on persisters is the enrichment of pure persisters. In this work, we validated a proposed method to isolate persisters from vancomycin and enrofloxacin generated persistent populations. With this, we analyzed the proteome profile of pure persisters and revealed the distinct mechanisms associated with vancomycin and enrofloxacin induced persisters. Furthermore, morphological and metabolic characterizations were performed, indicating further differences between these two persister populations. Finally, we assessed the effect of ATP repression, protein synthesis inhibition and reactive oxygen species (ROS) level on persister formation. In conclusion, this work provides a comprehensive understanding of S. aureus vancomycin and enrofloxacin induced persisters at the molecular, single cell and population levels, facilitating a better understanding of persisters and the development of effective strategies to combat them.

Lactobacillus acidophilus has been extensively applied in plentiful probiotic products and is mostly found in the gastrointestinal tract, vagina, and oral cavity of human and animal, and fermented foods. Although several studies have been performed to investigate the beneficial characteristics and genome function of L. acidophilus, comparative genomic analysis remains scarce. In this study, we collected 74 L. acidophilus genomes from our gut bacterial genome collection and the public database and conducted a comprehensive comparative genomic analysis. The analysis of average nucleotide identity (ANI), phylogenetic, gene distribution of COG and KEGG database, carbohydrates utilization, and secondary metabolites revealed the potential correlation of the genomic diversity and niches adaptation of L. acidophilus from different perspectives. In addition, the pan-genome of L. acidophilus was found to be open, with metabolism, information storage and processing genes mainly distributed in the core genome. Phage- and peptidase-associated genes were found in the genome of the specificity of animal-derived strains, which were related to adaptation of animal gut. SNP analysis showed the differences of the utilization of Vitamin B12 in cellular of L. acidophilus strains from animal gut and others. This work provides new insights for the genomic diversity analysis of Lactobacillus acidophilus and uncovers the ecological adaptation of the specific strains.

Radiation-resistant Deinococcus radiodurans is an extremophilic microorganism capable of withstanding high levels of ionizing radiation and chemical mutagens. It possesses remarkable DNA repair capability and serves as a model organism for studying stress resistance mechanism. However, our understanding on the spatial chromosome organization of this species remains limited. In this study, we employed chromosome conformation capture (3C) technology to determine the 3D genome structure of D. radiodurans and to further investigate the changes of chromosome conformation induced by ultraviolet (UV) irradiation. We observed that UV irradiation reduced short-range chromosome interactions, and smaller chromosomal interaction domains (CIDs) merged to form larger CIDs. Integrating transcriptomic data analysis, we found that the majority of upregulated differentially expressed genes were significantly enriched near specific CID boundaries. Specially, we comprehensively elucidated that the nucleoid-associated protein Dr_ebfC may serve as a global regulator for gene expression by altering chromosome structure, thereby influencing the physiological state of the bacterium. Overall, our study revealed the chromosome conformations of D. radiodurans under different conditions, and offered valuable insights into the molecular responses of this extremophile to environmental stresses.

Endozoicomonas, a core bacterial group in corals, may also be a coral symbiont. Endozoicomonas communities often decrease rapidly in corals under heat stress. However, how the bacteria respond to changes in temperature and coral host during heat stress is unknown. Here, we employed the cultivable, dominant species E. montiporae as a working organism to explore how Endozoicomonas responds to heat stress. We designed two experiments to clarify the extent to which E. montiporae is influenced by temperature and coral host. We detected differentially expressed protein (DEP) profiles in this bacterium at 31{degrees}C and 33{degrees}C compared to 25{degrees}C by tandem mass tags-based quantitative proteome analysis. Fifty DEPs, including many heat shock proteins, were detected when the temperature changed. The expression of antioxidant defense proteins and key pyruvate synthase proteins decreased, suggesting that E. montiporae were in a physiology of stress at 33{degrees}C. Furthermore, some proteins were differentially expressed because of the heat-stress-treated coral lysate specifically, suggesting that not only heat but also heat-induced host factors can affect the protein expression of the bacterium. This study provides an in-depth analysis of how the molecular mechanisms of Endozoicomonas are affected by heat stress and coral host.

Full genome sequences of seven mycobacteriophages isolated from environmental soil samples are presented. These bacteriophages, with their respective cluster or subclusters, are Duplo (A2), Dynamo (P1), Gilberta (A11), MaCh (A11), Nikao (K1), Phloss (N), and Skinny (M1). All were temperate Siphoviridae, with genome sizes ranging from 43,107-82,071 bp.

Diarrhea typically indicates an intestinal disorder, which can occur from viruses, parasites or bacterial infection. Along with the common diarrhea-causing pathogens, opportunistic bacteria may also play a role in the etiology of diarrheal disease. One of the opportunists bacteria that can cause diarrhea in both children and adults is Providencia stuartii. Therefore, the goal of this study is to explore the genetic mechanism of the opportunistic P. stuartii in microbial interactions with common diarrheal pathogens. Hence, P. stuartii was identified by utilizing the morphological observation and molecular techniques. Afterwards, the entire genome of P. stuartii was sequenced, assembled and annotated to explore the genomic insights. In addition, the virulence genes of 100 whole genome sequences from ten prevalent diarrhea-causing bacteria were identified and prioritized. Finally, the system biology approach was used to predict the protein-protein interaction network between P. stuartii and the virulence genes. The results of the present study suggests that complete genome sequencing of this bacteria contains 4011 proteins, which are crucial for this bacterium to survive. Additionally, 16 gene clusters provide 207 interacting genes that could interact with biological and molecular function, subcellular localization and pathway. The microbial interaction accompanying the virulence gene was found in all 10 diarrhea-causing bacteria except Clostridium difficile. These findings of this study could aid in the exploration of Providencia stuartii as the major causative agent of diarrhea. Additionally, the pathophysiology of diarrhea can be investigated using the microbial interactions between P. stuartii and the typical diarrheal bacteria. The results of this study may therefore be used to determine the most effective therapeutic targets for the development of medications to treat diarrhea.

Trillions of marine bacterial, archaeal and viral species contribute to the majority diversity of life on Earth. In the current study, we have done a comprehensive review of all the published studies of marine microbiome by re-analyzing most of the available high throughput sequencing data. We collected 17.59 Tb sequencing data from 8,165 metagenomic and prokaryotic samples, and systematically evaluated the genome characters, including genome size, GC content, phylogeny, and the functional and ecological roles of several typical phyla. A genome catalogue of 9,070 high quality genomes and a gene catalogue including 156,209,709 genes were constructed, representing the most integrate marine prokaryotic datasets till now. The genome size of Alphaproteobacteria and Actinobacteria was significant correlated to their GC content. A total of 44,322 biosynthetic gene clusters distributed in 53 types were detected from the reconstructed marine prokaryotic genome catalogue. Phylogenetic annotation of the 8,380 bacterial and 690 archaeal species revealed that most of the known bacterial phyla (99/111), including 62 classes and 181 orders, and four extra unclassified genomes from two candidate novel phyla were detected. In addition, taxonomically unclassified species represented a substantial fraction of 64.56% and 80.29% of the phylogenetic diversity of Bacteria and Archaea respectively. The genomic and ecological features of three groups of Cyanobacteria, luminous bacteria and methane-metabolizing archaea, including inhabitant preference, geolocation distribution and others were through discussed. Our database provides a comprehensive resource for marine microbiome, which would be a valuable reference for studies of marine life origination and evolution, ecology monitor and protection, bioactive compound development.

The unresponsiveness of bacterial tolerant subpopulations to antibiotics has been attributed to physiological dormancy triggered by environmental stresses. In this work, we showed that protein and DNA synthesis activities in tolerant subpopulations that formed during nutrient starvation remained at a high level and only dropped gradually during the course of six days-starvation. Interestingly, upon decline of these activities, the tolerant subpopulations became susceptible to antibiotics that target protein and DNA synthesis, which was found to be required to support DNA repair functions essential for prolonged survival of the tolerant subpopulation. The increasing susceptibility of the bacterial tolerant subpopulation to antibiotics was also due to diminishing energy production, antioxidant defense and efflux functions, which resulted in weakening defense and further inhibition of synthesis and functions of DNA repair and other defense proteins. These findings confirm that bacterial tolerant subpopulations remain physiological active, and open new opportunities for combating bacterial tolerance with existing antibiotics.

The peptidoglycan (PG; or murein) is a mesh-like structure, which is made of glycan polymers connected by short peptides and surrounds the cell membrane of nearly all bacterial species. In contrast, there is no PG counterpart that would be universally found in Archaea, but rather various polymers that are specific to some lineages. Methanopyrales and Methanobacteriales are two orders of Euryarchaeota that harbor pseudomurein (PM) in their cell-wall, a structural analogue of the bacterial PG. Owing to the differences between PG and PM biosynthesis, some have argued that the origin of both polymers is not connected. However, recents studies have revealed that the genomes of PM-containing Archaea encode homologues of the bacterial genes involved in PG biosynthesis, even though neither their specific functions nor the relationships within the corresponding inter-domain phylogenies have been investigated so far. In this work, we devised a bioinformatic pipeline to identify all potential proteins for PM biosynthesis in Archaea without relying on a candidate gene approach. After an in silico characterization of their functional domains, the taxonomic distribution and evolutionary relationships of the collected proteins were studied in detail in Archaea and Bacteria through HMM similarity searches and phylogenetic inference of the Mur domain-containing family, the ATP-grasp superfamily and the MraY-like family. Our results notably show that the extant archaeal muramyl ligases are ultimately of bacterial origin, but likely diversified through a mixture of horizontal gene transfer and gene duplication. Moreover, structural modeling of these enzymes allowed us to propose a tentative function for each of them in pentapeptide elongation. While our work clarifies the genetic determinants behind PM biosynthesis in Archaea, it also raises the question of the architecture of the cell wall in the last universal common ancestor.

In bacteria, cell division usually occurs through binary fission, with the participation of genes from the dcw cluster. In mollicutes, this cluster is significantly reduced -- often only the ftsZ, ftsA, mraZ, and mraW genes remain, and sometimes ftsZ is completely absent. FtsZ is a key division protein that forms the Z-ring, but its role in mollicutes is questionable due to the absence of many cell division proteins and the cell wall. In the current study, we investigated the FtsZ protein of Mycoplasma gallisepticum, a bacterium with a reduced set of putative cell division genes (ftsZ, ftsA, ftsK). The results show that, unlike in well-studied bacteria, FtsZ in M. gallisepticum often exhibits polar rather than mid-cell localization. Overexpression of fluorescently labeled FtsZ enhances this polar localization and may lead to minicell formation. The FtsZ concentration was measured and, together with in vitro data, confirmed its ability to polymerize, similar to its homologs. Protein-protein interactions were also analyzed and confirmed the link of FtsZ to cell division. Overall, the results support the role of FtsZ in cell division, though its properties differ significantly from other known homologs.