A new type of Gram-negative, aerobic, salt-tolerant, active, rod-shaped, and predatory bacteria ASxL5T was isolated from a cow dung pond in Nottinghamshire, England, and used Campylobacter as its prey. Subsequently, other Campylobacter species and members of the Enterobacteriaceae family were discovered as prey. After subculture without host cells, weak aseptic growth was achieved on Brain Heart Infusion Agar. The optimal growth conditions are 37 °C and pH is 7. Transmission electron microscopy revealed some very unusual morphological features related to the availability of prey. Phylogenetic analysis using the 16S rRNA gene sequence indicated that the isolate is related to a member of the Marine Spirulina family, but cannot be clearly classified as a member of any known genus. Whole-genome sequencing of ASxL5T confirmed the relationship with members of the marine spirochetes. A database search revealed that several ASxL5Ts share 16S rRNA gene sequences with several uncultured bacteria from the ocean, land surface and groundwater. We suggest that the strain ASxL5T represents a new species in a new genus. We recommend the name Venatorbacter cucullus gen. November, sp. In November, ASxL5T was used as the type strain.
Predatory bacteria are bacteria that exhibit the ability to hunt down and kill other living bacteria to obtain biosynthetic materials and energy. This is different from the general recovery of nutrients from dead microorganisms, and it is also different from parasitic interactions, in which bacteria form a close relationship with their host without killing them. Predatory bacteria have evolved different life cycles to take advantage of abundant food sources in the niches where they are found (such as marine habitats). They are a taxonomically diverse group, which are only connected by their unique sterilization life cycle1. Examples of predatory bacteria have been found in several different phyla, including: Proteobacteria, Bacteroides, and Chlorella.3. However, the most well-studied predatory bacteria are the Bdellovibrio and Bdellovibrio-and-like organisms (BALOs4). Predatory bacteria are a promising source of new biologically active compounds and antibacterial agents5.
Predatory bacteria are believed to enhance microbial diversity and have a positive impact on ecosystem health, productivity and stability6. Despite these positive attributes, there are few studies on new predatory bacteria due to the difficulty of culturing bacteria and the need to carefully observe cell interactions to understand their complex life cycles. This information is not easy to obtain from computer analysis.
In an era of increasing antimicrobial resistance, new strategies for targeting bacterial pathogens are being studied, such as the use of bacteriophages and predatory bacteria7,8. ASxL5T bacteria were isolated in 2019 using phage isolation technology from cow dung collected from the Dairy Center of the University of Nottingham, Nottinghamshire. The purpose of the investigation is to isolate organisms with potential as biological control agents. Campylobacter hyointestinalis is a zoonotic pathogen, which is increasingly associated with human intestinal diseases10. It is ubiquitous in serum and used as a target host.
The ASxL5T bacterium was isolated from beef jelly because it was observed that the plaques it formed on the lawn of C. hyointestinalis were similar to those produced by bacteriophages. This is an unexpected finding, because part of the phage isolation process involves filtering through a 0.2 µm filter, which is designed to remove bacterial cells. Microscopic examination of the material extracted from the plaque revealed that the small gram-negative curved rod-shaped bacteria did not accumulate polyhydroxybutyrate (PHB). The aseptic culture independent of prey cells is realized on rich solid medium (such as brain heart infusion agar (BHI) and blood agar (BA)), and its growth is weak. It is obtained after subculture with heavy inoculum improve. It grows equally well under microaerobic (7% v/v oxygen) and atmospheric oxygen conditions, but not in an anaerobic atmosphere. After 72 hours, the diameter of the colony was very small, reaching 2 mm, and it was beige, translucent, round, convex and shiny. Standard biochemical testing is hampered because ASxL5T cannot be cultured reliably in liquid media, which suggests that it may rely on the complex life cycle of biofilm formation. However, the plate suspension showed that ASxL5T is aerobic, positive for oxidase and catalase, and can tolerate 5% NaCl. ASxL5T is resistant to 10 µg streptomycin, but is sensitive to all other antibiotics tested. The ASxL5T bacterial cells were examined by TEM (Figure 1). When grown without prey cells on the BA, ASxL5T cells are small Campylobacter, with an average length of 1.63 μm (± 0.4), a width of 0.37 μm (± 0.08), and a single long (up to 5 μm) pole. Sexual flagella. Approximately 1.6% of the cells appear to have a width of less than 0.2 μm, which will allow passage through the filter device. An unusual structural extension was observed on the top of some cells, similar to a fairing (Latin cucullus) (see the arrows in 1D, E, G). This seems to be composed of excess outer membrane, which may be due to the rapid reduction in the size of the periplasmic envelope, while the outer membrane remains intact, showing a "loose" appearance. Culturing ASxL5T in the absence of nutrients (in PBS) for a long time at 4°C resulted in most (but not all) cells showing a coccal morphology (Figure 1C). When ASxL5T grows with Campylobacter jejuni as prey for 48 hours, the average cell size is significantly longer and narrower than cells grown without a host (Table 1 and Figure 1E). In contrast, when ASxL5T grows with E. coli as prey for 48 hours, the average cell size is longer and wider than when it grows without prey (Table 1), and the cell length is variable, usually showing filamentous (Figure 1F). When incubated with Campylobacter jejuni or E. coli as prey for 48 hours, ASxL5T cells showed no flagella at all. Table 1 summarizes the observations of changes in cell size based on the presence, absence, and prey type of ASxL5T.
TEM display of ASx5LT: (A) ASx5LT shows long whip; (B) typical ASx5LT battery; (C) cocci ASx5LT cells after long incubation without nutrients; (D) a group of ASx5LT cells show abnormality (E) ASx5LT cell group incubated with Campylobacter prey showed increased cell length compared with those without prey growth (D) also showed apical structure; (F) Large Filamentous flagella, ASx5LT cells, after incubation with E. coli prey; (G) A single ASx5LT cell after incubation with E. coli, showing an unusual top structure. The bar represents 1 μm.
Determining the 16S rRNA gene sequence (accession number MT636545.1) enables database searches to establish sequences similar to those in the Gammaproteobacteria class, and are closest to marine bacteria in the marine spirillum family (Figure 2), and are members of the Thalassolituus genus The closest relative to Marine Bacillus. The 16S rRNA gene sequence is clearly different from the predatory bacteria belonging to the Bdelvibrionaceae (Deltaproteobacteria) family. The pairwise comparisons of B. bacteriovorus HD100T (type strain, DSM 50701) and B. bacteriovorus DM11A were 48.4% and 47.7%, and for B. exovorus JSS it was 46.7%. ASxL5T bacteria have 3 copies of the 16S rRNA gene, two of which are identical to each other, and the third is 3 bases apart. Two other predatory bacterial isolates (ASx5S and ASx5O; 16S rRNA gene accession numbers are MT636546.1 and MT636547.1, respectively) with similar morphological and phenotypic characteristics from the same location are not the same, but they are different from ASxL5T and uncultured Bacterial database sequences are clustered together with other genera in Oceanospirillaceae (Figure 2). The whole genome sequence of ASxL5T has been determined and saved in the NCBI database, and the accession number is CP046056. The genome of ASxL5T consists of a circular chromosome of 2,831,152 bp with a G + C ratio of 56.1%. The genome sequence contains 2653 CDS (total), of which 2567 are predicted to encode proteins, of which 1596 can be assigned as putative functions (60.2%). The genome contains 67 RNA-coding genes, including 9 rRNAs (3 each for 5S, 16S, and 23S) and 57 tRNAs. The genomic characteristics of ASxL5T were compared with the available genomes of strains of the nearest relative type identified from the 16S rRNA gene sequence (Table 2). Use amino acid identity (AAI) to compare all available Thalassolituus genomes to ASxL5T. The closest available (incomplete) genome sequence determined by AAI is Thalassolituus sp. C2-1 (add NZ_VNIL01000001). This strain was isolated from the deep-sea sediments of the Mariana Trench, but there is currently no phenotypic information about this strain for comparison. Compared with ASxL5T's 2.82 Mb, the organism's genome is larger at 4.36 Mb. The average genome size of marine spirochetes is about 4.16 Mb (± 1.1; n = 92 complete reference genomes investigated from https://www.ncbi.nlm.nih.gov/assembly), so the genome of ASxL5T is in line with the order Compared to the other members, it is quite small. Use GToTree 1.5.54 to generate a genome-based estimated maximum likelihood phylogenetic tree (Figure 3A), using the aligned and linked amino acid sequences of 172 single-copy genes specific to Gammaproteobacteria 11,12,13,14,15,16, 17 ,18. The analysis showed that it is closely related to Thalassolituus, Bacterial Plane, and Marine Bacterium. However, these data indicate that ASxL5T is different from its relatives in Marine Spirulina and its genome sequence data is available.
The phylogenetic tree using the 16S rRNA gene sequence highlights the position of the ASxL5T, ASxO5, and ASxS5 strains (with the guts) relative to the uncultivated and marine bacteria strains in the Marine Spirulinaceae. The Genbank accession number follows the strain name in parentheses. Use ClustalW to align sequences, and use maximum likelihood method and Tamura-Nei model to infer phylogenetic relationships, and perform 1000 guided replications in the MEGA X program. The number on the branch indicates that the guided copy value is greater than 50%. Escherichia coli U/541T was used as an outgroup.
(A) A phylogenetic tree based on the genome, showing the relationship between the marine Spirospiraceae bacterium ASxL5T and its close relatives, E. coli U 5/41T as an outgroup. (B) Compared with T. oleivorans MIL-1T, the functional category distribution of genes is predicted based on the orthologous group (COG) cluster of ASx5LT protein. The figure on the left shows the number of genes in each functional COG category in each genome. The graph on the right shows the percentage of genomes contained in each functional COG group. (C) Compared with T. oleiverans MIL-1T, the analysis of the complete KEGG (Kyoto Encyclopedia of Genes and Genomes) modular pathway of ASxL5T.
Using the KEGG database to examine the component genes present in the ASxL5T genome revealed the typical metabolic pathway of aerobic gamma Proteus. ASxL5T contains a total of 75 genes assigned to bacterial motor proteins, including genes involved in chemotaxis, flagella assembly, and type IV fimbriae system. In the last category, 9 out of 10 genes are responsible for the twitching movement of a range of other organisms. The genome of ASxL5T contains a complete tetrahydropyrimidine biosynthetic pathway that participates in the protective response to osmotic stress20, as expected for halophiles. The genome also contains many complete pathways for cofactors and vitamins, including riboflavin synthesis pathways. Although the alkane 1-monooxygenase (alkB2) gene is present in ASxL5T, the hydrocarbon utilization pathway is not complete. In the genome sequence of ASxL5T, homologues of genes identified as mainly responsible for the degradation of hydrocarbons in T. oleiverans MIL-1T21, such as TOL_2658 (alkB) and TOL_2772 (alcohol dehydrogenase) are obviously absent. Figure 3B shows the comparison of gene distribution in the COG category between ASxL5T and Olive oil MIL-1T. Overall, the smaller ASxL5T genome contains proportionally fewer genes from each COG category compared to the larger related genome. When the number of genes in each functional category is expressed as a percentage of the genome, differences are noted in the percentage of genes in the translation, ribosomal structure and biogenesis categories, and the energy production and conversion function categories, which constitute the larger ASxL5T genome The percentage is compared with the same group present in the T. oleiverans MIL-1T genome. In contrast, compared with the ASxL5T genome, T. oleivorans MIL-1T has a higher percentage of genes in the replication, recombination and repair, and transcription categories. Interestingly, the biggest difference in the content of each functional category of the two genomes is the number of unknown genes present in ASxL5T (Figure 3B). An enrichment analysis of KEGG modules was performed, where each KEGG module represents a set of manually defined functional units for annotation and biological interpretation of genome sequence data. The comparison of gene distribution in the complete KOG module pathway of ASxL5T and olive MIL-1T is shown in Figure 3C. This analysis shows that although ASxL5T has a complete sulfur and nitrogen metabolic pathway, T. oleiverans MIL-1T does not. In contrast, T. oleiverans MIL-1T has a complete cysteine ​​and methionine metabolic pathway, but it is incomplete in ASxL5T. Therefore, ASxL5T has a characteristic module for sulfate assimilation (defined as a set of genes that can be used as phenotypic markers, such as metabolic capacity or pathogenicity; https://www.genome.jp/kegg/module.html) In T. oleiverans MIL-1T. Comparing the gene content of ASxL5T with the list of genes that suggest a predatory lifestyle is inconclusive. Although the waaL gene encoding the ligase associated with the O antigen polysaccharide to the core is present in the ASxL5T genome (but it is common in many Gram-negative bacteria), tryptophan 2,3-dioxygenase (TDO ) Genes may include the 60 amino acid regions commonly found in predatory bacteria that are not present. There are no other predatory characteristic genes in the ASxL5T genome, including those encoding enzymes involved in isoprenoid biosynthesis in the mevalonate pathway. Note that there is no transcriptional regulatory gene gntR in the predator group examined, but three gntR-like genes can be identified in ASxL5T.
The phenotypic characteristics of ASxL5T are summarized in Table 3 and compared with the phenotypic characteristics of related genera 23, 24, 25, 26, and 27 reported in the literature. Isolates from T. marinus, T. olevorans, B. sanyensis, and Oceanobacter kriegii are active, salt-tolerant, oxidase-positive rod-shaped bodies, but have almost no other phenotypic characteristics with ASxL5T. The average pH of the ocean is 8.1 (https://ocean.si.edu/ocean-life/invertebrates/ocean-acidification#section_77), which is reflected in T. marinus, T. olevorans, B. sanyensis and O. kriegii. ASxL5T is suitable for the larger pH range (4-9) typical of non-marine species. Phenotypic characteristics of Thalassolituus sp. C2-1. Unknown. The growth temperature range of ASxL5T is generally wider than that of marine strains (4–42 °C), although some but not all T. marinus isolates are heat-tolerant. The inability to grow ASxL5T in broth media prevented further phenotypic characterization. Use API 20E to test the materials scraped from the BA plate, ONPG, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, urease, tryptophan deaminase, gelatin hydrolysis Enzyme, the test results were all negative, but no indole, acetoin and H2S were produced. Unfermented carbohydrates include: glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. Compared with the published related reference strains, the cellular fatty acid profile of the ASxL5T strain is shown in Table 4. The main cellular fatty acids are C16:1ω6c and/or C16:1ω7c, C16:0 and C18:1ω9. Hydroxy fatty acids C12:0 3-OH and C10:0 3-OH also exist. The ratio of C16:0 in ASxL5T is higher than the reported value of related genera. In contrast, compared with the reported T. marinus IMCC1826TT, the ratio of C18:1ω7c and/or C18:1ω6c in ASxL5T is reduced. oleivorans MIL-1T and O. kriegii DSM 6294T, but not detected in B. sanyensis KCTC 32220T. Comparing the fatty acid profiles of ASxL5T and ASxLS revealed subtle differences in the amount of individual fatty acids between the two strains, which are consistent with the genomic DNA sequence of the same species. No poly-3-hydroxybutyrate (PHB) particles were detected using the Sudan black test.
The predation activity of ASxL5T bacteria was studied to determine the range of prey. This bacterium can form plaques on Campylobacter species, including: Campylobacter suis 11608T, Campylobacter jejuni PT14, Campylobacter jejuni 12662, Campylobacter jejuni NCTC 11168T; Escherichia coli NCTC 12667; C. helveticus NCTC 12472; C lari NCTC 11458 and C. upsaliensis NCTC 11541T. Use the cultures listed in the host range determination section of the method to test a wider range of Gram-negative and Gram-positive bacteria. The results show that ASxL5T can also be used in Escherichia coli NCTC 86 and Citrobacter freundii NCTC 9750T. Plaques formed on Klebsiella oxytoca 11466. The TEM interaction with E. coli NCTC 86 is shown in Figure 4A-D, and the interaction with Campylobacter jejuni PT14 and Campylobacter suis S12 is shown in Figure 4E-H middle. The attack mechanism seems to be different between the prey types tested, with one or more E. coli cells attached to each ASxL5T cell and positioned laterally along the extended cell before adsorption. In contrast, ASxL5T appears to attach to Campylobacter through a single point of contact, usually in contact with the apex of the predator cell and near the apex of the Campylobacter cell (Figure 4H).
TEM showing the interaction between ASx5LT and prey: (AD) and E. coli prey; (EH) and C. jejuni prey. (A) A typical ASx5LT cell connected to a single E. coli (EC) cell; (B) A filamentous ASx5LT attached to a single EC cell; (C) A filamentous ASx5LT cell connected to multiple EC cells; (D) Attachment Smaller ASx5LT cells on a single E. coli (EC) cell; (E) a single ASx5LT cell connected to a Campylobacter jejuni (CJ) cell; (F) ASx5LT attacks C. hyointestinalis (CH) cells; (G) two One ASx5LT cell attacked a CJ cell; (H) A close-up view of the ASx5LT attachment point, near the apex of the CJ cell (bar 0.2 μm). The bar represents 1 μm in (A–G).
Predatory bacteria have evolved to take advantage of abundant sources of prey. Obviously, they are widely present in many different environments. Due to the narrow size of the population members, it is possible to isolate ASxL5T bacteria from the slurry using the phage separation method. The genomic relevance of ASxL5T to members of the oceanospirillaceae family of marine bacteria is surprising, although the organism is salt-tolerant and can grow on a medium containing 5% salt. Water quality analysis of the slurry showed that the sodium chloride content was less than 0.1%. Therefore, mud is far away from the marine environment-both geographically and chemically. The presence of three related but different isolates from the same source provides evidence that these predators are thriving in this non-marine environment. In addition, microbiome analysis (data files available from https://www.ebi.ac.uk/ena/browser/view/PRJEB38990) showed that the same 16S rRNA gene sequence is located in the top 50 most abundant operational taxa (OTU ) In a few sampling intervals of the mud. Several uncultured bacteria were found in the Genbank database, which have 16S rRNA gene sequences similar to ASxL5T bacteria. These sequences, together with the sequences of ASxL5T, ASxS5, and ASxO5, seem to represent different clades separated from Thalassolituus and Oceanobacter (Figure 2). Three kinds of uncultured bacteria (GQ921362, GQ921357 and GQ921396) were isolated from the fissure water at a depth of 1.3 kilometers in the South African gold mine in 2009, and the other two (DQ256320 and DQ337006) were from groundwater (also in South Africa in 2005). The 16S rRNA gene sequence most closely related to ASxL5T is part of the 16S rRNA gene sequence obtained from the enrichment culture of sandy sediments obtained from the beaches of northern France in 2006 (accession number AM29240828). Another closely related 16S rRNA gene sequence from the uncultured bacterium HQ183822.1 was obtained from a collection tank leached from a municipal landfill in China. Obviously, ASxL5T bacteria are not highly representative in taxonomic databases, but these sequences from uncultured bacteria are likely to represent organisms similar to ASxL5T, which are distributed all over the world, usually in challenging environments. From the whole genome phylogenetic analysis, the closest relative to ASxL5T is Thalassolituus sp. C2-1, T. marinus, T. oleivorans. And O. kriegii 23, 24, 25, 26, 27. Thalassolituus is a member of marine obligate hydrocarbon fragmentation bacteria (OHCB), which is widespread in marine and terrestrial environments, and usually becomes the dominant after hydrocarbon pollution incidents30,31. Marine bacteria are not members of the OHCB group, but are isolated from the marine environment.
Phenotypic data indicate that ASxL5T is a new species and a member of a previously unrecognized genus in the marine spirospiraceae family. There is currently no clear standard to classify newly isolated strains into a new genus. Attempts have been made to determine universal genera boundaries, for example, based on the percentage of the genome of a conservative protein (POCP), it is recommended that the cut-off value is 50% identical to the reference strain33. Others suggest using AAI values, which have advantages over POCP because they can be obtained from incomplete genomes34. The author believes that if the AAI value is less than 74% compared with the model strain of the model species, the strain is a representative of a different genera. The model genus in the marine spirillaceae is marine spirillum, and the model strain is O. linum ATCC 11336T. The AAI value between ASxL5T and O. linum ATCC 11336T is 54.34%, and the AAI value between ASxL5T and T. oleivorans MIL-1T (genus type strains) is 67.61%, indicating that ASxL5T represents a new genus different from Thalassolituus. Using the 16S rRNA gene sequence as the classification standard, the suggested genus delimitation boundary is 94.5%35. ASxL5T may be placed in the Thalassolituus genus, showing 95.03% 16S rRNA sequence identity with T. oleivorans MIL-1T and 96.17%. marinus IMCC1826T. However, it will also be placed in the Bacteroides genus that has 94.64% 16S rRNA gene identity with B. sanyensis NV9, indicating that the use of a single gene such as the 16S rRNA gene can lead to arbitrary classification and assignment. Another suggested method uses ANI and Genome Alignment Score (AF) to examine the clustering of data points from all types and non-type strains of existing genera. The author recommends combining the genus boundary with the inflection point of the estimated genus specific to the taxa being analyzed. However, if there are not enough complete genome sequences from Thalassolituus isolates, it is impossible to determine whether ASxL5T belongs to the Thalassolituus genus by this method. Due to the limited availability of complete genome sequences for analysis, the entire genome phylogenetic tree should be interpreted with caution. Secondly, whole genome comparison methods cannot account for substantial differences in the size of the compared genomes. They measured the similarity of conserved core single-copy genes between related genera, but did not take into account the large number of genes that are not present in the much smaller genome of ASxL5T. Obviously, ASxL5T and groups including Thalassolituus, Oceanobacter, and Bacterioplanes have a common ancestor, but evolution has taken a different path, leading to a reduction in the genome, which may be to adapt to a predatory lifestyle. This is in contrast to T. oleivorans MIL-1T, which is 28% larger and has evolved under different environmental pressures to utilize hydrocarbons23,30. An interesting comparison can be made with obligate intracellular parasites and symbionts, such as Rickettsia, Chlamydia, and Buchnera. Their genome size is about 1 Mb. The ability to utilize host cell metabolites leads to gene loss, so Underwent significant evolutionary genomic degradation. Evolutionary changes from marine chemical nutrient organisms to predatory lifestyles may result in a similar reduction in genome size. The COG and KEGG analysis highlights the number of genes used for specific functions and the global differences in the genomic pathways between ASxL5T and T. oleivorans MIL-1T, which are not due to the widespread availability of mobile genetic elements. The difference in the G + C ratio of the whole genome of ASxL5T is 56.1%, and that of T. oleivorans MIL-1T is 46.6%, which also indicates that it is segregated.
Examination of the coding content of the ASxL5T genome provides functional insights into phenotypic characteristics. The presence of genes encoding type IV fimbriae (Tfp) is of particular interest because they promote cell movement, called social gliding or convulsions, without flagella on the surface. According to reports, Tfp has other functions, including predation, pathogenesis, biofilm formation, natural DNA uptake, automatic cell aggregation and development38. The ASxL5T genome contains 18 genes encoding diguanylate cyclase (an enzyme that catalyzes the conversion of 2 guanosine triphosphate into guanosine 2 phosphate and cyclic diGMP) and 6 genes encoding the corresponding diguanylate cyclase phosphate diguanylate. The gene for esterase (catalyzing the degradation of cyclic di-GMP to guanosine monophosphate) is interesting because cycl-di-GMP is an important second messenger involved in biofilm development and separation, movement, cell attachment and virulence 39, 40 in the process. It should also be noted that in Bdellovibrio bacteriovorus, cyclic double GMP has been shown to control the transition between free life and predatory lifestyle41.
Most research on predatory bacteria has focused on Bdellovibrio, Bdellovibrio-like organisms, and Myxococcus species. These and other known examples of predatory bacteria form a diverse group. Despite this diversity, a set of characteristic protein families that reflect the phenotypes of 11 known predatory bacteria have been identified3,22. However, only genes encoding O antigen ligase (waaL) have been identified, which is particularly common in Gram-negative bacteria. This form of analysis is not helpful in designating ASxL5T as a predator, probably because it uses a novel attack strategy. The availability of more diverse predatory bacterial genomes will help develop finer resolution analyses that take into account evidence of functional and environmental differences between group members. Examples of predatory bacteria not included in this analysis include members of Cupriavidus necator42 and Bradymonabacteria43, because as researchers investigate different microbial communities, more predatory taxa are established.
The most notable feature of ASxL5T bacteria captured by TEM image is its unique and flexible morphology, which can promote interaction with prey bacteria. The type of interaction observed is different from other predatory bacteria and has not been previously discovered or reported. The proposed ASxL5T predatory life cycle is shown in Figure 5. There are few examples in the literature with similar apical structures as we report here, but these examples include Terasakiispira papahanaumokuakeensis, a marine spirillum bacterium with occasional apex enlargement 44, and Alphaproteobacteria, Terasakiella pusilla, formerly belonging to the genus Oceanospirillum, exhibiting The so-called "polar film" 45. Cocci forms are often observed in older cultures, especially for bacteria with curved forms, such as Vibrio, Campylobacter, and Helicobacter 46, 47, 48, which may represent a degraded state. Further work is needed to clarify the precise life cycle of ASxL5T bacteria. To determine how it captures and preys, and whether its genome encodes biologically active compounds that can be used for medical or biotechnological purposes.
Description of Venatorbacter gen. November Venatorbacter (Ven.a.tor, ba'c.ter, L. is composed of venators from L. n. venator,'hunter' and Gr. n. bacter,'a rod'. Venatorbacter,'a hunting Rod'. Cells are aerobic, salt-tolerant, curved Gram stain negative, exercise rod. Catalase and oxidase activity are positive. PHB does not accumulate. In the temperature range of 4 to 42 °C Ingrown. The pH range of 4-9 is unusual. In marine snails, most are intolerant of acidic pH. The main fatty acids are C16:1ω6c and/or C16:1ω7c, C16:0 and C18:1ω9 ; C12:0 3-OH and C10:0 3-OH are found as hydroxy fatty acids. They do not grow in broth media. The DNA G + C content is 56.1 mol%. Members of this genus show resistance to Campylobacter And the predation behavior of members of the Enterobacteriaceae family. The phylogenetic position of this genus is in the family.
Description of Venatorbacter cucullus sp. November Venatorbacter cucullus (cu'cull.us.; L. n. cucullus means fairing).
In addition, the descriptive feature of this genus is that when grown on BA or BHI, the cells are 1.63 µm long and 0.37 µm wide. The colonies on BHI agar are very small, reaching 2 mm in diameter after 72 hours. They are beige, translucent, round, convex and shiny. The members of this species can use Escherichia coli and Klebsiella. Campylobacter and several other Gram-negative bacteria serve as prey.
The typical strain ASxL5T was isolated from beef milk in Nottinghamshire, UK, and deposited in the National Type Culture Collection (UK): accession number NCTC 14397 and the Netherlands Bacterial Culture Collection (NCCB) accession number NCCB 100775. The complete genome sequence of ASxL5T has been deposited in Genbank according to the addition of CP046056.
ASxL5T bacteria were isolated from beef milk using phage isolation technology9,49. The slurry was diluted 1:9 (w/v) in SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O and 0.01% gelatin; Sigma Aldrich, Gillingham, UK), Then incubate at 4°C for 24 hours, rotating slowly to elute the predators into the buffer. The suspension was centrifuged at 3000g for 3 minutes. The supernatant was collected and centrifuged at 13,000g for a second time for 5 minutes. The supernatant was then passed through a 0.45 µm membrane filter (Minisart; Sartorius, Gottingen, Germany) and a 0.2 µm membrane filter (Minisart) to remove any remaining bacterial cells. ASxL5T can pass these filters. A soft agar lawn of Campylobacter enterosus S12 (NCBI accession number CP040464) from the same slurry was prepared using standard techniques. The filtered slurry was distributed on each of these host cell plates in 10 µl droplets in triplicate and allowed to dry. The plate was incubated in a microaerophilic tank at 37°C for 48 hours under microaerobic conditions (5% O2, 5% H2, 10% CO2, and 80% N2). The obtained visible plaque was extracted into SM buffer and transferred to the fresh lawn of C. hyointestinalis S12 to further propagate the lysed organisms. Once it is determined that the bacteria are the cause of the lytic plaque and not the phage, try to grow the organism independently of the host and further characterize it. The aerobic culture was performed at 37 °C with 5% v/v defibrinated horse blood (TCS Biosciences Lt, Buckingham, UK, supplement). According to the guidelines of the National Clinical Standards Committee, the disc diffusion method is used for antibacterial susceptibility testing. BHI agar was cultured at 37 °C using a disc containing the following antibiotics (Oxoid) for aerobic culture: amoxicillin and clavulanic acid 30 µg; cefotaxime 30 µg; streptomycin 10 µg; ciprofloxacin 5 µg; Ceftazidime 30 µg Nalidixic acid 30 µg; Imipenem 10 µg; Azithromycin 15 µg; Chloramphenicol 30 µg; Cefoxitin 30 µg; Tetracycline 30 µg; Nitrofurantoin 300 µg; Aztreonam 30 µg; Ampicillin 10 µg ; Cefpodoxime 10 µg; Trimethoprim-Sulfamethoxazole 25 µg. The salt tolerance was established by aerobic incubation on BHI agar plates at 37 °C. Additional NaCl was added to the BHI agar plates to provide a concentration range of up to 10% w/v. The pH range is determined by aerobic culture on BHI agar plates at 37°C, where the pH range has been adjusted to between 4 and 9 with sterile HCl or sterile NaOH, and the target pH value is verified before pouring the plate . For cellular fatty acid analysis, ASxL5T was cultured on BHI agar for 3 days and aerobic at 37 °C. According to the MIDI (Sherlock Microbial Identification System, version 6.10) standard protocol of FERA Science Ltd, (York, UK), cell fatty acids were extracted, prepared and analyzed.
For TEM, ASxL5T was cultured aerobic by spreading uniformly on BA at 37°C for 24 hours, and then harvested into 1 ml of 3% (v/v) glutaraldehyde in 0.1 M cacodylate buffer at room temperature Fix for 1 hour, then centrifuge at 10,000 g for 3 minutes. Then gently resuspend the pellet in 600 μl 0.1 M cacodylate buffer. Transfer the fixed ASxL5T suspension to the Formvar/carbon film on a 200 mesh copper grid. The bacteria were stained with 0.5% (w/v) uranyl acetate for 1 minute and examined by TEM using a TEI Tecnai G2 12 Biotwin microscope. As mentioned above, combine the same number of prey and predator in NZCYM broth (BD Difco™, Fisher Scientific UK Ltd, Loughborough) and incubate for 48 hours under microaerobic conditions of Campylobacter or Campylobacter at 37°C , The interaction of predator and prey was also examined by TEM. Aerobic conditions for Escherichia coli. Independently examine prey and predatory bacteria to determine any changes in cell morphology due to predation. The Sudan black method was used for optical microscopy of PHB accumulation.
Grow ASxL5T overnight cultures by smearing growth on BHI or BA plates with a sterile swab. Collect ASxL5T cells and suspend them in MRD (CM0733, Oxoid), and then place them at 4°C for 7 days to starve the cells. The NCTC reference or laboratory stock bacterial culture was inoculated into BHI broth or No. 2 nutrient broth (CM007, Oxoid), incubated overnight, centrifuged at 13,000g and resuspended in MRD until OD600 was 0.4. Culture: Bacillus subtilis NCTC 3610T, Citrobacter freundii NCTC 9750T, Enterobacter aerogenes NCTC 10006T, Enterococcus faecalis NCTC 775T, Escherichia coli NCTC 86, Klebsiella oxytoca 11466, Leuconostoc NCTC 10817, Listeria Special bacteria NCTC 4885, Bacillus macerans NCTC 6355T, Providencia stuartsii NCTC 10318, Pseudomonas fluorescens SMDL, Rhodococcus submarine hamburger NCTC 1621T, Salmonella intestinal bacteria Mondeville NCTC 5747, mucilage NCTC 10861, Staphylococcus aureus NCTC 8532T, Streptococcus pneumoniae NCTC 7465T, Yersinia enterocolitica NCTC 10460. The Campylobacter host was microaerobically incubated on BA plates at 37°C and suspended in NZCYM broth. The tested Campylobacter hosts are: C. coli 12667 NCTC, C. jejuni 12662, C. jejuni PT14, C. jejuni NCTC 11168T, C. helveticus NCTC 12472, C. lari NCTC 11458, C. lari NCTC 11458, C. jejuni PT14, C... Collect cells in MRD, centrifuge at 13,000g and resuspend in MRD until OD600 is 0.4. Add an aliquot of 0.5 ml suspension to 5 ml melted NZCYM top agar (0.6% agar) and pour it onto a 1.2% NZCYM bottom plate. After curing and drying, the serially diluted ASxL5T was distributed as 20 µl droplets on each lawn board in triplicate. The culture temperature and atmosphere depend on the requirements of the test bacteria.
Use GenElute™ Bacterial Genomic DNA Kit (Sigma Aldridge) to prepare DNA from bacterial isolates. Standard methods were used for PCR amplification of 16S rRNA gene and product sequence determination using dye termination chemistry (Eurofins Value Read Service, Germany). Use the BLAST-N program to compare these sequences with other 16S rRNA gene sequences to identify and collect closely related species. These are aligned using ClustalW in the MEGA X program. The phylogenetic tree was reconstructed using MEGA X using the maximum likelihood method based on the Tamura-Nei model, with 1000 guided copies54. Use PureLink™ Genomic DNA Kit (Fisher Scientific, Loughborough, UK) to extract DNA for whole-genome sequencing. The genome sequence of ASxL5T was determined using the Illumina MiSeq combination, which consists of 250 bp double-ended reads composed of a library prepared using the Nextera labeling kit and 2 to 20 kb long reads from the PacBio platform. Genomics DNA Sequencing Research Facility at Sembia University. The genome was assembled using CLC Genomics Workbench 12.0.3 (Qiagen, Aarhus, Denmark). ASxL5T cultures are deposited in the National Type Culture Collection (UK) and the Netherlands Bacterial Culture Collection (NCCB). The genomes of related organisms used for comparison are: Thalassolituus oleivorans MIL-1T (accession number HF680312, complete); Bacterioplanes sanyensis KCTC 32220T (accession number BMYY01000001, incomplete); Oceanobacter kriegii DSM 6294T (accession number NZ_AUGV00000000, incomplete); Marinamonas community DSM 5604T (added ASM436330v1, incomplete), Oceanospirullum linum ATCC 11336T (added MTSD02000001, incomplete) and Thalassolituus sp. C2-1 (add NZ_VNIL01000001, incomplete). Use JGI Genome Portal36 at https://img.jgi.doe.gov//cgi-bin/mer/main.cgi?section=ANI&page= to determine the alignment score (AF) and average nucleic acid identity (ANI). In pairs. The method of Rodriguez-R & Konstantinidis55 was used to determine amino acid identity (AAI). Use GToTree 1.5.5411,12,13,14,15,16,17,18 to generate an estimated maximum likelihood phylogenetic tree. The input genome representing the available reference genome is selected from reference genera identified as being related to ASxL5T from the 16S rRNA phylogeny. Annotated the tree using the interactive tree of life online tool (https://itol.embl.de/). The functional annotation and analysis of the ASxL5T genome is carried out using the BlastKOALA KEGG online tool using the KEGG (Kyoto Encyclopedia of Genes and Genomes) module enrichment distribution. The distribution of COG categories (orthologous groups) is determined using the eggNOG-mapper online tool.
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