Bacillus Phage Research Articles

Dynamics of bacteriophages in gut of giant pandas reveal a potential regulation of dietary intake on bacteriophage composition

Fecal samples of cubs and adults of giant pandas were examined to determine the effects of diets on the diversity and dynamics of gut bacteriophages. Enterobacteria phage, Salmonella phage, Escherichia phage, Shigella phage, Klebsiella phage, and Lactococcus phage were found to be dominant in both cub and adult samples. Citrobacter phage, Cronobacter phage, Pectobacterium phage, Erwinia phage, Dickeya phage, Erwinia phage, Enterococcus phage, and Pseudomonas phage were more abundant in adults, while Lactococcus phage, Streptococcus phage, Lactobacillus phage, and Leuconostoc phage were more abundant in cubs. The abundance and diversity of the majority of phage species were increased in pandas with age. There was an increase in the abundance of Pectobacterium phage and a decrease in the abundance of Lactobacillus phage, Leuconostoc phage, Bacillus phage, and Streptococcus phage in adults. As cubs and adults of giant pandas have different dietary habits, these observations suggest a significant effect of diets on the composition and abundance of gut bacteriophages in giant pandas.

Phage vB_BmeM-Goe8 infecting Bacillus megaterium DSM319.

vB_BmeM-Goe8 is a phage preying on Bacillus megaterium. Its genome has a GC content of 38.9%, is 161,583bp in size, and has defined ends consisting of 7436-bp-long terminal repeats. It harbours 11 genes encoding tRNAs and 246 coding DNA sequences, 66 of which were annotated. The particle reveals Myoviridae morphology, and the formation of a double baseplate upon tail sheath contraction indicates morphological relatedness to the group of SPO1-like phages. BLASTn comparison against the NCBI non-redundant nucleotide database revealed that Bacillus phage Mater is the closest relative of vB_BmeM-Goe8.

Genome Sequences of Two Bacillus Phages Isolated from Indonesia.

Here, the genome sequences of two soil bacteriophages isolated from a red chili plantation in Indonesia are presented. The genome of vB_BspS_SplendidRed (42,859 bp) is highly similar to Bacillus phage Ray17 from the United States, while vB_BspM_MarvelLand (156,945 bp) is highly similar to Bacillus phage BC01 from South Korea.

Characterization of Bacillus phage FrankLy and genomic analysis of Mycobacterium phage phreeze for use in phage-antibiotic therapy

Characterization of Bacillus phage FrankLy and genomic analysis of Mycobacterium phage phreeze for use in phage-antibiotic therapy

A68 Characterization and comparative genomic analysis of two Bacillus megaterium lytic bacteriophages

Next-generation sequencing technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Many Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. Here, we describe biological characteristics and whole-genome sequences and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), isolated from Egyptian soil samples and representing two different groups according to their host range and amplified fragment length polymorphism profiles. Both phages have been displaying different thermal inactivation points (82 and 78 °C) and pH tolerance range (5–9.2 and 5–8.4 pH) while having the same longevity in vitro (192 h). Electron microscopy observation has proved that both phages belonged to the Myoviridiae family. Furthermore, growth analyses indicated that phages BM5 and BM10 have a shorter latent period (20 and 25 min) and smaller burst size (103 and 117 PFU) than is typical for Bacillus phages. The genome sizes of phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of BM5 and BM10 genomes enabled assignments of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of BM5 and BM10 genome structures with other B. megaterium bacteriophages revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7) within L cluster and display different lifestyles (lysogenic and lytic). Sequenced phages encode proteins associated with Bacillus pathogenesis. BM5 does not contain any tRNA sequences, while BM10 genome codes for 17 tRNAs.

Caffeine, ethanol, and genomic factors influence Bacillus and Gordonia phage life cycle decisions

Caffeine, ethanol, and genomic factors influence Bacillus and Gordonia phage life cycle decisions

Bacillus Phage vB_BtS_B83 Previously Designated as a Plasmid May Represent a New Siphoviridae Genus.

The Bacillus cereus group of bacteria includes, inter alia, the species known to be associated with human diseases and food poisoning. Here, we describe the Bacillus phage vB_BtS_B83 (abbreviated as B83) infecting the species of this group. Transmission electron microscopy (TEM) micrographs indicate that B83 belongs to the Siphoviridae family. B83 is a temperate phage using an arbitrium system for the regulation of the lysis–lysogeny switch, and is probably capable of forming a circular plasmid prophage. Comparative analysis shows that it has been previously sequenced, but was mistaken for a plasmid. B83 shares common genome organization and >46% of proteins with other the Bacillus phage, BMBtp14. Phylograms constructed using large terminase subunits and a pan-genome presence–absence matrix show that these phages form a clade distinct from the closest viruses. Based on the above, we propose the creation of a new genus named Bembunaquatrovirus that includes B83 and BMBtp14.

Structural insights into DNA recognition by AimR of the arbitrium communication system in the SPbeta phage

A newly identified arbitrium communication system regulates the lysis-to-lysogeny decision in a Bacillus bacteriophage. This system contains an arbitrium hexapeptide as a signal, the cellular receptor AimR, and the lysogenic negative regulator AimX. AimR specifically targets the downstream DNA to activate aimX gene expression. The arbitrium peptide binds to AimR, inhibiting its DNA-binding to promote phage lysogeny. Recently, we and other groups have elucidated how arbitrium peptide sensed by AimR. However, the molecular mechanisms of DNA recognition by AimR and the regulation of its DNA-binding activity by the peptide remain largely unknown. Here, we report the crystal structure of the AimR–DNA complex at 2.1 Å resolution. The N-terminal HTH motif recognizes the palindromic DNA sequence, buttressed by interactions between positively charged residues and the DNA phosphate groups. The DNA-bound AimR assembles a more closed dimer than the peptide-bound form. Single-molecule FRET and crosslinking assays revealed that the AimR protein samples both open and closed conformations in solution. Arbitrium peptide binding induces a closed-to-open conformational change of AimR, eliminating DNA targeting. Our structural and functional analysis provides new insights into the DNA recognition mechanism of AimR and its regulation by the arbitrium peptide in the context of phage lysis-lysogeny decisions.

Complete Genome Sequences of Bacillus Bacteriophages Wes44 and Carmen17.

Wes44 and Carmen17 are siphoviruses that infect Bacillus thuringiensis DSM-350. Wes44 contains 42,248 base pairs and 54 predicted genes; Carmen17 contains 41,820 base pairs and 51 predicted genes. The genomes are 95% similar to each other and distantly related to Bacillus cereus bacteriophage PBC1.

Complete genome sequence of the virus isolate vB_BthM-Goe5 infecting Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is non-pathogenic for humans and serves as a biological control agent in agriculture. Understanding its phages will help to prevent industrial production loss of Bt products and will lead to a better understanding of phages in general. The complete genome of the new B. thuringiensis phage isolate vB_BthM-Goe5 (Goe5) was sequenced, revealing a linear 157,804-bp-long dsDNA chromosome flanked by 2579-bp-long terminal repeats. It contains two tRNAs and 272 protein coding regions, 69 of which could be assigned with an annotation. Morphological investigation, using transmission electron microscopy, revealed Myoviridae morphology. The formation of a double baseplate upon tail sheath contraction indicates a link to the group of SPO1-related phages. Comparative genomics with all Bacillus-related viral genomes available in the NCBI genome database during this investigation indicated that Goe5 was a unique isolate, with Bacillus phage Bastille as its closest relative.

Inactivation of health-related microorganisms in water using UV light-emitting diodes

Abstract UV light-emitting diodes (UV-LEDs) offer various wavelength options, while microorganisms have spectral sensitivity, or so-called action spectra, which can be different among species. Accordingly, matching properly the emission spectra of UV-LEDs and the spectral sensitivity of microorganisms is a reasonable strategy to enhance inactivation. In this study, UV-LEDs with nominal peak emissions at 265, 280 and 300 nm were applied to pathogens including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus and feline calicivirus, in comparison with indicator species including Escherichia coli, Bacillus subtilis spores, bacteriophage Qβ and MS2. The results indicated that, for all species tested, 265 nm UV-LED was highest in the fluence-based inactivation rate constant k, followed by 280 nm and 300 nm was much lower. The k value at 280 nm was close to that at 265 nm for feline calicivirus and MS2, suggesting that 280 nm UV-LED can be as good an option as 265 nm UV-LED to inactivate these viruses. Bacteria tended to show fluence-response curves with shoulder and tailing, while viruses followed log-linear profiles at all wavelengths tested. This study indicates the fluence-response profiles and the fluence required for a target inactivation of microorganisms, which would serve as reference data for future study and applications of UV-LEDs.

Phage Reduce Stability for Regaining Infectivity during Antagonistic Coevolution with Host Bacterium.

The coevolution between phage and host bacterium is an important force that drives the evolution of the microbial community, yet the coevolution mechanisms have still not been well analyzed. Here, by analyzing the interaction between a Bacillus phage vB_BthS_BMBphi and its host bacterium, the coevolution mechanisms of the first-generation phage-resistant bacterial mutants and regained-infectivity phage mutants were studied. The phage-resistant bacterial mutants showed several conserved mutations as a potential reason for acquiring phage resistance, including the mutation in flagellum synthesis protein FlhA and cell wall polysaccharide synthesis protein DltC. All the phage-resistant bacterial mutants showed a deleted first transmembrane domain of the flagellum synthesis protein FlhA. Meanwhile, the regain-infectivity phage mutants all contained mutations in three baseplate-associated phage tail proteins by one nucleotide, respectively. A polymorphism analysis of the three mutant nucleotides in the wild-type phage revealed that the mutations existed before the interaction of the phage and the bacterium, while the wild-type phage could not infect the phage-resistant bacterial mutants, which might be because the synchronized mutations of the three nucleotides were essential for regaining infectivity. This study for the first time revealed that the synergism mutation of three phage baseplate-associated proteins were essential for the phages’ regained infectivity. Although the phage mutants regained infectivity, their storage stability was decreased and the infectivity against the phage-resistant bacterial mutants was reduced, suggesting the phage realized the continuation of the species by way of “dying to survive”.

Isolation and Characterization of A Bacillus Phage from Equine Carcass Disposal Site

Bacteriophages play an important role in bacterial control in natural niche however a little is known about Bacillus sp. phages prevailing in cadaver affected soils. In the current study, the Bacillus sp. phage was isolated from the equine cadaver disposal site and characterised to gain an insight into the issue of role of phages in biological dynamics of manure thus formed over years. Firstly, the host bacterium was isolated and identified as Bacillus cereus group member as assessed by phylogenetic analysis and secondly it’s corresponding phage from same soil sample was also enriched and characterised. The phage (VTCCBPA38) was found to belong to family Myoviridae and was active within the temperature range of 4°C - 45°C. As assessed by biological sensitivity by spot test, the phage was active against 6/19 (31.6 %) Bacilli tested including Bacillus cereus from goat mastitis. Thus the phagemay find potential use in biocontrol of diseases caused by Bacillus sp. Furthermore, this report is valuable as the first study for investigation of Bacillus sp. phage in carcass burial sites.

Characterization and comparative genomic analysis of virulent and temperate Bacillus megaterium bacteriophages.

Next-Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Several Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 min, respectively) and a smaller burst size (103 and 117 PFU, respectively), in comparison to what is typical for Bacillus phages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with other B. megaterium bacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7, respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic, respectively). Moreover, sequenced phages encode proteins associated with Bacillus pathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin.

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Bacillus safensis FO-36b and Bacillus pumilus SAFR-032: a whole genome comparison of two spacecraft assembly facility isolates

BackgroundBacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilus ATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis.ResultsThe FO-36b gene order is essentially the same as that in SAFR-032 and other B. pumilus strains. The annotated genome has 3850 open reading frames and 40 noncoding RNAs and riboswitches. Of these, 307 are not shared by SAFR-032, and 65 are also not shared by MERTA and ATCC7061T. The FO-36b genome has ten unique open reading frames and two phage-like regions, homologous to the Bacillus bacteriophage SPP1 and Brevibacillus phage Jimmer1. Differing remnants of the Jimmer1 phage are found in essentially all B. safensis / B. pumilus strains. Seven unique genes are part of these phage elements. Whole Genome Phylogenetic Analysis of the B. pumilus, B. safensis and other Firmicutes genomes, separate them into three distinct clusters. Two clusters are subgroups of B. pumilus while one houses all the B. safensis strains. The Genome-genome distance analysis and a phylogenetic analysis of gyrA sequences corroborated these results.ConclusionsIt is not immediately obvious that the presence or absence of any specific gene or combination of genes is responsible for the variations in resistance seen. It is quite possible that distinctions in gene regulation can alter the expression levels of key proteins thereby changing the organism’s resistance properties without gain or loss of a particular gene. What is clear is that phage elements contribute significantly to genome variability. Multiple genome comparison indicates that many strains named as B. pumilus likely belong to the B. safensis group.

Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens

Aim:Present investigation was conducted to isolate and characterize bacteriophages with lytic activity against common bacterial pathogens.Materials and Methods:A total of 60 samples of animal waste disposal from cattle (42) and buffalo (18) farms were collected from three different strata, i.e., top, mid, and bottom of collection tank. Samples were primarily subjected to rapid detection methods, and then isolation of phage was done by double agar layer method using Bacillus subtilis (BsH) and Escherichia coli (EH) as host system. Phages were characterized on the basis of plaque morphology, temperature, pH susceptibility, and host range.Results:Recovery of phages was higher from dairy cattle farm waste (78.57%) as compared to buffalo farm waste (72.22%) and bottom layer of tank showed maximum recovery. Bacillus subtilis (91%) supported the growth of more phages as compared to E. coli (9%). Three different phage morphotypes were observed each against Bacillus subtilis (BsHR1, BsHR2, and BsHR3) and E. coli (EHR1, EHR2, and EHR3). Mean phage titer of above six phage isolates ranged between 3×1010 and 5×1012 plaque forming units/ml. Viability of phages was by, and large unaffected at 70°C within 2-3 min, and phage isolates were completely inactivated below pH 3 and above 11. Coliphage EHR1 had widest host range followed by BsHR1 and BsHR2 while EHR2, EHR3, and BsHR3 had low lytic activity.Conclusion:It could be concluded from the present study that the Bacillus and Coli phage has wide host range and thus exhibits the potential to be used as drug substitute tool against common bacterial pathogens.

Predicting genome terminus sequences of Bacillus cereus-group bacteriophage using next generation sequencing data

BackgroundMost tailed bacteriophages (phages) feature linear dsDNA genomes. Characterizing novel phages requires an understanding of complete genome sequences, including the definition of genome physical ends.ResultWe sequenced 48 Bacillus cereus phage isolates and analyzed Next-generation sequencing (NGS) data to resolve the genome configuration of these novel phages. Most assembled contigs featured reads that mapped to both contig ends and formed circularized contigs. Independent assemblies of 31 nearly identical I48-like Bacillus phage isolates allowed us to observe that the assembly programs tended to produce random cleavage on circularized contigs. However, currently available assemblers were not capable of reporting the underlying phage genome configuration from sequence data. To identify the genome configuration of sequenced phage in silico, a terminus prediction method was developed by means of ‘neighboring coverage ratios’ and ‘read edge frequencies’ from read alignment files. Termini were confirmed by primer walking and supported by phylogenetic inference of large DNA terminase protein sequences.ConclusionsThe Terminus package using phage NGS data along with the contig circularity could efficiently identify the proximal positions of phage genome terminus. Complete phage genome sequences allow a proposed characterization of the potential packaging mechanisms and more precise genome annotation.

Bacteriophages of soil bacilli: A new multivalent phage of Bacillus altitudinis

Bacillus are soil saprophytes, facultative anaerobes developing in the temperature range of 28–37°С. 16S rRNA cataloging shows that these bacteria form a coherent class with broad variability of virulence. Bacillus phages can be extensively used for phagotyping bacteria in the process of soil, water, and food monitoring. Bacillus phages can also be used as vectors in horizontal gene transfer and potential therapeutic agents. Thus, description of the biological diversity of the Bacillus phages is useful for further development of tools used in molecular biology and biomedicine. In this work, the scheme for isolation of soil bacteriophages was unified, which allowed ten bacillus phages to be isolated from different types of soil. It was shown that the number of phages depended on the soil fertility, decreasing as the soil changed from black soil to chestnut soil to gray forest soil to uncontaminated urban soil to oil-contaminated urban soil. A new polyvalent DNA-containing bacteriophage SRT01hs of B. altitudinis (it is also able to infect B. subtilis, B. cereus, and B. pumilus, but not B. licheniformis and B. atrophaeus) was described in detail. It has a typical structure: a total length of 360 nm and an icosahedron-shaped head 100 nm in diameter. Several phages simultaneously attack a B. altitudinis cell by increasing the level of intracellular low-molecular RNA. Infection with the phage virtually eliminates the stationary growth phase of infected bacilli and leads to a permanent increase in the number of phages in cultural liquor, with the exception of the time period of high activity of the secreted ribonuclease.