The genome of Symbiodiniaceae-associated Stutzerimonas frequens CAM01 reveals a broad spectrum of antibiotic resistance genes indicating anthropogenic drift in the Palk Bay coral reef of south-eastern India.

An increase in antibiotic pollution in reef areas will lead to the emergence of antibiotic-resistant bacteria, leading to ecological disturbances in the sensitive coral holobiont. This study provides insights into the genome of antibiotics-resistant Stutzerimonas frequens CAM01, isolated from Favites-associated Symbiodiniaceae of a near-shore polluted reef of Palk Bay, India. The draft genome contains 4.67 Mbp in size with 52 contigs. Further genome analysis revealed the presence of four antibiotic-resistant genes, namely, adeF, rsmA, APH (3")-Ib, and APH (6)-Id that provide resistance by encoding resistance-nodulation-cell division (RND) antibiotic efflux pump and aminoglycoside phosphotransferase. The isolate showed resistance against 73% of the antibiotics tested, concurrent with the predicted AMR genes. Four secondary metabolites, namely Aryl polyene, NRPS-independent-siderophore, terpenes, and ectoine were detected in the isolate, which may play a role in virulence and pathogenicity adaptation in microbes. This study provides key insights into the genome of Stutzerimonas frequens CAM01 and highlights the emergence of antibiotic-resistant bacteria in coral reef ecosystems.

Gut microbiome responses in the metabolism of human dietary components: Implications in health and homeostasis.

The gut microbiome and its link with human health and disease have gained a lot of attention recently. The microbiome executes its functions in the host by carrying out the transformation of dietary components and/or de novo synthesis of various essential nutrients. The presence of complex microbial communities makes it difficult to understand the host-microbiome interplay in the metabolism of dietary components. This review attempts to uncover the incredible role of the gut microbiome in the metabolism of dietary components, diet-microbiome interplay, and restoration of the microbiome. The in silico analysis performed in this study elucidates the functional description of essential/hub genes involved in the amino acid degradation pathway, which are mutually present in the host and its gut microbiome. Hence, the computational model helps comprehend the inter-and intracellular molecular networks between humans and their microbial partners.

A novel anti-infective molecule nesfactin identified from sponge associated bacteria Nesterenkonia sp. MSA31 against multidrug resistant Pseudomonas aeruginosa.

Overuse of antibiotics coupled with biofilm-forming ability has led to the emergence of multi-drug P. aeruginosa strains worldwide. Quorum sensing is a bacterial cell-cell communication system that regulates the expression of genes, including virulence factors, through production of acyl-homoserine lactones (AHLs) in Pseudomonas aeruginosa. The phenotypic expression of virulence factors in P. aeruginosa is mediated by quorum sensing systems (las and rhl). In this study an anti-infective molecule produced by a marine actinomycetes Nesterenkonia sp. MSA31 was elucidated as lipopeptide by NMR and LC-MS/MS analysis. The new lipopeptide molecule was named Nesfactin. This molecule effectively inhibited virulence phenotypes including production of hemolysin, protease, lipase, phospholipase, esterase, elastase, rhamnolipid, alginate, and pyocyanin, as well as motility and biofilm formation in P. aeruginosa. The high-performance thin layer chromatography (HPTLC) analysis revealed that the lipopeptide (50 µg/mL) inhibited production of the AHLs produced by the las and rhl quorum sensing systems (3-oxo-C12-HSL and C4-HSL, respectively). Docking analysis showed the binding affinity of the ligand towards the quorum sensing receptor molecules. The confocal laser scanning microscopy images showed the anti-biofilm effect of lipopeptide against P. aeruginosa. Nesfactin based hydrogel showed a significant antibiofilm effect on the catheter. This study suggests that the lipopeptide may be an effective anti-virulence treatment for Pseudomonas aeruginosa infections.

A hijack mechanism of Indian SARS-CoV-2 isolates for relapsing contemporary antiviral therapeutics.

Coronavirus disease (COVID-19) rapidly expands to a global pandemic and its impact on public health varies from country to country. It is caused by a new virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is imperative for relapsing current antiviral therapeutics owing to randomized genetic drift in global SARS-CoV-2 isolates. A molecular mechanism behind the emerging genomic variants is not yet understood for the prioritization of selective antivirals. The present computational study was aimed to repurpose existing antivirals for Indian SARS-CoV-2 isolates by uncovering a hijack mechanism based on structural and functional characteristics of protein variants. Forty-one protein mutations were identified in 12 Indian SARS-CoV-2 isolates by analysis of genome variations across 460 genome sequences obtained from 30 geographic sites in India. Two unique mutations such as W6152R and N5928H found in exonuclease of Surat (GBRC275b) and Gandhinagar (GBRC239) isolates. We report for the first time the impact of folding rate on stabilizing/retaining a sequence-structure-function-virulence link of emerging protein variants leading to accommodate hijack ability from current antivirals. Binding affinity analysis revealed the effect of point mutations on virus infectivity and the drug-escaping efficiency of Indian isolates. Emodin and artinemol suggested herein as repurposable antivirals for the treatment of COVID-19 patients infected with Indian isolates. Our study concludes that a protein folding rate is a key structural and evolutionary determinant to enhance the receptor-binding specificity and ensure hijack ability from the prevalent antiviral therapeutics.

A gelatinized lipopeptide diet effectively modulates immune response, disease resistance and gut microbiome in Penaeus vannamei challenged with Vibrio parahaemolyticus.

Penaeus vannamei is one of the most economically vital shrimp globally, but infectious diseases have hampered its proper production and supply. As antibiotics pose a huge threat to the environment and humankind, it is essential to seek an alternative strategy to overcome infection and ensure proper culture and production. The present study investigates the effect of an anti-infective biosurfactant derivative lipopeptide MSA31 produced by a marine bacterium on the growth performance, disease resistance, and the gut microbiome of P. vannamei when challenged with pathogenic Vibrio parahaemolyticus SF14. The shrimp were fed with a commercial and lipopeptide formulated diet for 60 days and the growth performance was analyzed. The lipopeptide fed shrimp group showed enhanced growth performance and specific growth rate with improved weight gain than the control group. The challenge experiment showed that the survival rate was significant in the lipopeptide fed group compared to the control group. The results revealed 100% mortality in the control group at the end of 12 h of challenge, while 50% of the lipopeptide diet-fed group survived 24 h, which indicates the enhanced disease resistance in shrimp fed with a lipopeptide diet. The test group also showed higher levels of digestive and immune enzymes, which suggests that the lipopeptide diet could positively modulate the digestive and immune activity of the shrimp. The gut microbiome profiling by Illumina high-throughput sequencing revealed that the most abundant genera in the lipopeptide diet-fed group were Adhaeribacter, Acidothermus, Brevibacillus, Candidatus, Mycobacterium, Rodopila, and Streptomyces, while opportunistic pathogens such as Streptococcus, Escherichia, Klebsiella, Neisseria, Rhizobium, and Salmonella were abundant in the control diet-fed shrimp. Also, lipopeptide diet-fed shrimp were found to have a high abundance of ammonia and nitrogen oxidizing bacteria, which are essential pollutant degraders. Therefore, the study reveals that the dietary supplementation of lipopeptide in shrimp aquaculture could positively modulate the gut microbiome and enhance the shrimp's overall health and immunity in an eco-friendly manner.

Zoonotic evolution and implications of microbiome in viral transmission and infection.

The outbreak and spread of new strains of coronavirus (SARS-CoV-2) remain a global threat with increasing cases in affected countries. The evolutionary tree of SARS-CoV-2 revealed that Porcine Reproductive and Respiratory Syndrome virus 2, which belongs to the Beta arterivirus genus from the Arteriviridae family is possibly the most ancient ancestral origin of SARS-CoV-2 and other Coronaviridae. This review focuses on phylogenomic distribution and evolutionary lineage of zoonotic viral cross-species transmission of the Coronaviridae family and the implications of bat microbiome in zoonotic viral transmission and infection. The review also casts light on the role of the human microbiome in predicting and controlling viral infections. The significance of microbiome-mediated interventions in the treatment of viral infections is also discussed. Finally, the importance of synthetic viruses in the study of viral evolution and transmission is highlighted.

Phylogenomic proximity and comparative proteomic analysis of SARS-CoV-2.

The coronavirus disease (COVID-19) belongs to the family Severe Acute Respiratory Syndrome (SARS-CoV). It can be more severe for some persons and can lead to pneumonia or breathing difficulties resulting in the death of immune-compromised patients. We performed a phylogenomic and phylogeographic tree from the collected datasets. Phylogenomic analysis or sequence-based phylogeny showed an evolutionary relationship between the geographical strains. The phylogenomic tree grouped into two major clades consists of various isolates of SARS-CoV-2 and Bat SARS-like coronavirus, Bat coronavirus, and Pangolin coronavirus. The phylogenetic neighbor of newly sequenced Indian strains (Accession: MT012098.1, MT050493.1) was revealed to identify the variations between the nCoV-19 strains. The results showed keen evidence that SARS-CoV-2 has evolved from Bat SARS-like coronavirus. The evolutionary history and comparative proteomic analysis provide a new avenue for the current scientific research related to the coronavirus.

Comparative genomic analysis reveals starvation survival systems in Methanothermobacter thermautotrophicus ΔH.

Methanothermobacter thermautotrophicus ΔH (MTH) is a thermophilic hydrogenotrophic methanogenic archaeon capable of reducing CO2 with H2 to produce methane gas. It is the potential candidate in the biomethanation of CO2 and CO in anaerobic reactors and biogas upgrading process. However, systematic studies addressing its genome conservation and function remain scant in this genome. In this study, we have evaluated its evolutionary resemblance and metabolic discrepancy, particularly in starvation survival systems by comparing the genomic contexts with Methanothermobacter marburgensis str. Marburg (MMG) and Methanobacterium formicicum DSM 1535 (MFO). The phylogenomic analysis of this study indicated that there was a strong phylogenomic signal among MTH, MMG, and MFO in the whole-genome tree. DNA replication machinery was conserved in the MTH genome and might have evolved at different evolution rates. Genome synteny analysis observed collinearity of either gene orders or gene families has to be maintained with syntenic blocks located in the syntenic out-paralogs. A genome-wide metabolic analysis identified some unique putative metabolic subsystems in MTH, which are proposed to determine its growth characteristics in diverse environments. MTH genome comprised of 93 unique genes-coding for starvation survival and stress-response proteins. These proteins confer its adaptation to nutritional deprivation and other abiotic stresses. MTH has a typical system to withstand its growth and cell viability during stable operation and recovery after prolonged starvation. Thus, the present work will provide an insight to improve the genome refinement and metabolic reconstruction in parallel to other closely related species.

Functional prediction, characterization, and categorization of operome from Acetoanaerobium sticklandii DSM 519.

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia producing anaerobic bacterium that can be able utilizes amino acids as sole carbon and energy sources for its growth and energetic metabolism. A lack of knowledge on its molecular machinery and 30.5% conserved hypothetical proteins (HPs; operome) hinders the successful utility in biofuel applications. In this study, we have predicted, characterized and categorized its operome whose functions are still not determined accurately using a combined bioinformatics approach. The functions of 64 of the 359 predicted HPs are involved in diverse metabolic subsystems. A. sticklandii operome has consisted of 16% Rossmann fold and 46% miscellaneous folds. Subsystems-based technology has classified 51 HPs contributing to the small-molecular reactions, 26 in macromolecular reactions and 12 in the biosynthesis of cofactors, prosthetic groups and electron carriers. A generality of functions predicted from its operome contributed to the cell cycle, amino acid metabolism, membrane transport, and regulatory processes. Many of them have duplicated functions as paralogs in this genome. A. sticklandii has the ability to compete with invading microorganisms and tolerate abiotic stresses, which can be overwhelmed by the predicted functions of its operome. Results of this study revealed that it has specialized systems for amino acid catabolism-directed solventogenesis and acidogenesis but the level of gene expression may determine the metabolic function in amino acid fermenting niches in the rumina of cattle. As shown by our analysis, the predicted functions of its operome allow us for a better understanding of the growth and physiology at systems-scale.

Deciphering Molecular Virulence Mechanism of Mycobacterium tuberculosis Dop isopeptidase Based on Its Sequence-Structure-Function Linkage.

The pupylation pathway marks proteins for prokaryotic ubiquitin-like protein (Pup)-proteasomal degradation and survival strategy of mycobacteria inside of the host macrophages. Deamidase of Pup (Dop) plays a central role in the pupylation pathway. It is still a matter of investigation to know the function of Dop in virulence of mycobacterial lineage. Hence, the present study was intended to describe the sequence-structure-function-virulence link of Dop for understanding the molecular virulence mechanism of Mycobacterium tuberculosis H37Rv (Mtb). Phylogenetic analysis of this study indicated that Dop has extensively diverged across the proteasome-harboring bacteria. The functional part of Dop was converged across the pathogenic mycobacterial lineage. The genome-wide analysis pointed out that the pupylation gene locus was identical to each other, but its genome neighborhood differed from species to species. Molecular modeling and dynamic studies proved that the predicted structure of Mtb Dop was energetically stable and low conformational freedom. Moreover, evolutionary constraints in Mtb Dop were intensively analyzed for inferring its sequence-structure-function relationships for the full virulence of Mtb. It indicated that evolutionary optimization was extensively required to stabilize its local structural environment at the side chains of mutable residues. The sequence-structure-function-virulence link of Dop might have retained in Mtb by reordering hydrophobic and hydrogen bonding patterns in the local structural environment. Thus, the results of our study provide a quest to understand the molecular virulence and pathogenesis mechanisms of Mtb during the infection process.

Substrate-imprinted docking of Agrobacterium tumefaciens uronate dehydrogenase for increased substrate selectivity.

Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) belongs to the short-chain dehydrogenase superfamily, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. It is apparently required for the production of D-glucaric acid. AtuUdh-catalyzed reaction is reversible with dual substrate-specific activity (D-galacturonic acid and D-glucuronic acid) in nature. In our study, 34 mutants were pre-screened from 155 mutants generated from AtuUdh (wild-type) and selected 10 structurally stable mutants with increased substrate selectivity. The specificity, efficiency, and selectivity of these mutants for different substrates and cofactors were predicted from 121 docked models using a substrate-imprinted docking approach. Q14F, S36L, and S75T mutants have shown a high binding affinity to D-glucuronic acid and its substrate intermediates such as D-glucaro-1,4-lactone and D-glucaro-1,5-lactone. These mutants exhibited a low binding affinity to the substrate and cofactor required for D-galactaric acid. D34S, N112E and S165E mutants found to show a high selectivity of D-galacturonic acid and its substrate intermediates for D-galactaric acid production. Ser75, Ser165, and Arg174 are active residues playing an imperative role in the substrate selectivity and also contributed in the conjecture the mechanism of transition state stabilization catalyzed by AtuUdh mutants. The present approach was used to reveal the substrate binding mechanism of AtuUdh mutants for a better understanding of the structural basis for selectivity and function.

Deciphering structure, function and mechanism of Plasmodium IspD homologs from their evolutionary imprints.

Malaria is a life-threatening mosquito-borne blood disease caused by infection with Plasmodium parasites. Anti-malarial drug resistance is a global threat to control and eliminate malaria and therefore, it is very important to discover and evaluate new drug targets. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) homolog is a second in vivo target for fosmidomycin within isoprenoid biosynthesis in malarial parasites. In the present study, we have deciphered the sequence-structure-function integrity of IspD homologs based on their evolutionary imprints. The function and catalytic mechanism of them were also intensively studied by using sequence-structure homology, molecular modeling, and docking approach. Results of our study indicated that substrate-binding and dimer interface motifs in their structures were extensively conserved and part of them closely related to eubacterial origins. Amino acid substitutions in their coiled-coil regions found to bring a radical change in secondary structural elements, which in turn may change the local structural environment. Arg or Asp was identified as a catalytic site in plasmodium IspD homologs, contributing a direct role in the cytidylyltransferase activity similar to bacterial IspD. Results of molecular docking studies demonstrated how anti-malarial drugs such as fosmidomycin and FR-900098 have competitively interacted with the substrate-binding site of these homologs. As shown by our analysis, species-specific evolutionary imprints in these homologs determine the sequence-structure-function-virulence integrity and binding site alterations in order to confer anti-malarial drug resistance.

Functional annotation of operome from Methanothermobacter thermautotrophicus ΔH: An insight to metabolic gap filling.

Methanothermobacter thermautotrophicus ΔH (MTH) is a potential methanogen known to reduce CO2 with H2 for producing methane biofuel in thermophilic digesters. The genome of this organism contains ~50.5% conserved hypothetical proteins (HPs; operome) whose function is still not determined precisely. Here, we employed a combined bioinformatics approach to annotate a precise function to HPs and categorize them as enzymes, binding proteins, and transport proteins. Results of our study show that 315 (35.6%) HPs have exhibited well-defined functions contributing imperative roles in diverse cellular metabolism. Some of them are responsible for stress-response mechanisms and cell cycle, membrane transport, and regulatory processes. The genome-neighborhood analysis found five important gene clusters (dsr, ehb, kaiC, cmr, and gas) involving in the energetic metabolism and defense systems. MTH operome contains 223 enzymes with 15 metabolic subsystems, 15 cell cycle proteins, 17 transcriptional regulators and 33 binding proteins. Functional annotation of its operome is thus more fundamental to a profound understanding of the molecular and cellular machinery at systems-level.

Molecular evolution and functional divergence of IspD homologs in malarial parasites.

Malaria is one of the leading parasitic diseases to humans caused by Plasmodium falciparum. It is imperative to discover novel targets for the development of antimalarial drugs. The 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) in 2-C-methyl-D-erythritol-4-phosphate pathway has been considered as a second in vivo off-target for antimalarial drugs discovery as its essentiality in malarial parasites and devoid in mammals. Our study was intended to reveal the molecular basis of its functional parts by inferring diversity, origin and evolution across important malarial parasites. Phylogenetic analyses revealed its conservation probability and sequence homology among bacterial IspD homologs. It also indicated that Plasmodium IspD homologs were distantly related to each other and their functional counterparts originated from different progenitor genes. Nucleotide-diphospho-sugar transferase fold and conserved domain of them might have evolved from green sulphur bacteria, whereas coiled-coil region and apicoplast targeting signal derived from protozoal origins. These homologs contained prospectively definable motifs subject to neutral or nearly neutral evolution on a scale that were diverged radically and subsequently converged in making spatial structural arrangements. Our genetic diversity analysis has shown a constructive signal for identifying the evolutionary constraints, which has imposed on their functional divergence in malarial parasites. Thus, this study provides a novel insight into our understanding of the molecular basis of the origin and evolution history of IspD homologs across apicomplexa.

Methanobacterium formicicum as a target rumen methanogen for the development of new methane mitigation interventions: A review.

Methanobacterium formicicum (Methanobacteriaceae family) is an endosymbiotic methanogenic Archaean found in the digestive tracts of ruminants and elsewhere. It has been significantly implicated in global CH4 emission during enteric fermentation processes. In this review, we discuss current genomic and metabolic aspects of this microorganism for the purpose of the discovery of novel veterinary therapeutics. This microorganism encompasses a typical H2 scavenging system, which facilitates a metabolic symbiosis across the H2 producing cellulolytic bacteria and fumarate reducing bacteria. To date, five genome-scale metabolic models (iAF692, iMG746, iMB745, iVS941 and iMM518) have been developed. These metabolic reconstructions revealed the cellular and metabolic behaviors of methanogenic archaea. The characteristics of its symbiotic behavior and metabolic crosstalk with competitive rumen anaerobes support understanding of the physiological function and metabolic fate of shared metabolites in the rumen ecosystem. Thus, systems biological characterization of this microorganism may provide a new insight to realize its metabolic significance for the development of a healthy microbiota in ruminants. An in-depth knowledge of this microorganism may allow us to ensure a long term sustainability of ruminant-based agriculture.

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

Structural constraints-based evaluation of immunogenic avirulent toxins from Clostridium botulinum C2 and C3 toxins as subunit vaccines.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 and C3 toxins in addition to botulinum neurotoxins in avian and mammalian cells. C2 and C3 toxins are members of bacterial ADP-ribosyltransferase superfamily, which modify the eukaryotic cell surface proteins by ADP-ribosylation reaction. Herein, the mutant proteins with lack of catalytic and pore forming function derived from C2 (C2I and C2II) and C3 toxins were computationally evaluated to understand their structure-function integrity. We have chosen many structural constraints including local structural environment, folding process, backbone conformation, conformational dynamic sub-space, NAD-binding specificity and antigenic determinants for screening of suitable avirulent toxins. A total of 20 avirulent mutants were identified out of 23 mutants, which were experimentally produced by site-directed mutagenesis. No changes in secondary structural elements in particular to α-helices and ß-sheets and also in fold rate of all-ß classes. Structural stability was maintained by reordered hydrophobic and hydrogen bonding patterns. Molecular dynamic studies suggested that coupled mutations may restrain the binding affinity to NAD(+) or protein substrate upon structural destabilization. Avirulent toxins of this study have stable energetic backbone conformation with a common blue print of folding process. Molecular docking studies revealed that avirulent mutants formed more favorable hydrogen bonding with the side-chain of amino acids near to conserved NAD-binding core, despite of restraining NAD-binding specificity. Thus, structural constraints in the avirulent toxins would determine their immunogenic nature for the prioritization of protein-based subunit vaccine/immunogens to avian and veterinary animals infected with C. botulinum.

Structure-function discrepancy in Clostridium botulinum C3 toxin for its rational prioritization as a subunit vaccine.

Clostridium botulinum is anaerobic pathogenic bacterium causing food-born botulism in human and animals by producing botulinum neurotoxins A-H, C2, and C3 cytotoxins. Physiological group III strains (type C and D) of this bacterium are capable of producing C2 and C3 toxins in cattle and avian. Herein, we have revealed the structure-function disparity of C3 toxins from two different C. botulinum type C phage (CboC) and type D phage (CboD) to design avirulent toxins rationally. Structure-function discrepancy of the both toxins was computationally evaluated from their homology models based on the conservation in sequence-structure-function relationships upon covariation and point mutations. It has shown that 8 avirulent mutants were generated from CboC of 34 mutants while 27 avirulent mutants resulted from CboD mutants. No major changes were found in tertiary structure of these toxins; however, some structural variations appeared in the coiled and loop regions. Correlated mutation on the first residue would disorder or revolutionize the hydrogen bonding pattern of the coevolved pairs. It suggested that the residues coupling in the local structural environments were compensated with coevolved pairs so as to preserve a pseudocatalytic function in the avirulent mutants. Avirulent mutants of C3 toxins have shown a stable structure with a common blue print of folding process and also attained a near-native backrub ensemble. Thus, we concluded that selecting the site-directed mutagenesis sites are very important criteria for designing avirulent toxins, in development of rational subunit vaccines, to cattle and avian, but the vaccine specificity can be determined by the C3 toxins of C. botulinum harboring phages.