Heads or tails? An insight into the nature of antibacterial proteins of entomopathogenic bacteria.

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N.Key Points• Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified• It defined the autocidal activity of Linocin M18 (encapsulating) protein• Identified the possible killing mechanism of the encapsulins

The world of phage tail-like bacteriocins: State of the art and biotechnological perspectives.

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Nigerian ecosystem has been subjected to destruction by petroleum product spillage and other effluents resulting from operational activities of man with little or no regard to aquatic environment. These effluents' constituents have severe toxicological consequences for both the aquatic environment and the people hence a growing interest in exploiting the use of ecofriendly biodegradation approach. The aim of this research is to carry out a comparative chromatographic study of two bacteria species, Alcaligenes faecalis and Lysinibacillus sphaericus. The bacteria species were isolated from polluted Okulu River in Rivers State and characterized by molecular technique. Four treatment options were set up labelled A to D using samples from the polluted water body. Thus; Control (A), Alcaligenes faecalis(B) Lysinibacillus sphaericus (C) Alcaligenes faecalis + Lysinibacillus sphaericus (D). Each of the biodegradation experimental set-ups was incubated at room temperature for 42 days and total bacterial count and total petroleum hydrocarbon measured forthnightly. At end of 42 days, the result showed more activity with the mixed culture (99.4 %) followed by Lysinibacillus sphaericus (94.8 %) and Alcaligenes faecalis with 89.3 % reduction rate. The ability to utilize hydrocarbon is evident by the isolates used in this research. Total bacteria count at the end of 42 days were Alcaligenes faecalis (1.7 x 107), Lysinibacillus sphaericus (2.2 x 106) and mixed culture (2.0 x 107). The increasing growth during degradation was attributed to their hydrocarbon assimilating capabilities or adaptation of the strains due to their previous exposure to hydrocarbon in the river. From the result obtained in this research, these microbes have shown hydrocarbon degrading ability and can be used to clean up sites polluted with petroleum hydrocarbon.

Biopesticides, using living microbial bodies and their bio-active composites against insects, are potential replacements for synthetic insecticides for safer and modern food production systems. Entomopathogenic bacteria (EPB) are important biological control agents of insect pests since the last century. Though bacterial species have been documented to be used against insects for developing symbiotic relationships, only a few of them are identified as entomopathogens. Most of these are members of the family Bacillaceae, Enterobacteriaceae, Pseudomonadaceae, Clostridiaceae, and Neisseriaceae. More than 100 bacterial species have been reported to infect various arthropods. Bacillus thuringiensis (Bt), B. sphaericus, B. cereus, and B. popilliae are the most appreciated microbial pest control agents. However, new bacterial species also need to be explored for their entomopathogenic role and materialized as new biopesticide products. The commercial biopesticides based on novel EPBs with improved genetic materials must be a part of future research for effective integrated pest management programs. This present chapter highlights the classification, infection, replication, transmission mechanisms, and important EPB in integrated pest management.KeywordsEntomopathogenic bacteriaEPBBacillus spp.IPMBiopesticides

Aphids (Hemiptera: Aphididae) are cosmopolitan generalist pests of many agricultural crops. Their ability to reproduce rapidly through parthenogenesis allows them to quickly reach population sizes that are difficult to control. Their damage potential is further exacerbated when they act as vectors for plant pathogens, causing diseases in plants. Aphids are typically managed through the widespread use of insecticides, increasing the likelihood of short-term insecticide resistance. However, for the past few decades, entomopathogenic bacteria have been used as an alternative management strategy. Entomopathogenic bacteria have demonstrated their effectiveness for biologically suppressing insect pests, including aphids. In addition to identifying bacterial species that are pathogenic to aphids, research has been conducted on toxins such as Cry, Cyt, Vip, recombinant proteins, and other secondary metabolites with insecticidal activity. Most studies on aphids have been conducted in vitro, exposing them to an artificial diet contaminated with entomopathogenic bacteria or bacterial metabolites for periods ranging from 24 to 96 h. The discovery of new bacterial species with insecticidal potential, as well as the possibility of biotechnological applications through the genetic improvement of crops, will provide more alternatives for managing these agricultural pests in the future. This will also help address challenges related to field application.

BackgroundBacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics.ResultsUsing a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature.ConclusionThis study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Occurrence of protein phosphorylation in various bacterial species

This study describes the antibacterial activity of the pure isolates from Piper longum (L.) (black pepper) and Taxus baccata (L.) (Yew). Three isolates of black pepper were active against Gram-positive bacteria and moderately active against Gram-negative bacteria. Each isolate was highly active against at least one particular species of bacteria; piperlonguminine (1) against Bacillus subtilis, piperine (2) against Staphylococcus aureus, and pellitorine (3) against Bacillus sphaericus. 3-(3'-4'-5'-Trimethoxyphenyl) propionicacid (4) did not show any antibacterial activity. The isolate (--)-rhododendrol (5) of Taxus baccata (L.) inhibited Salmonella typhimurium and Pseudomonas syringae, while 4-(4'-hydroxyphenyl)-butan-2-one (6) and 4-(4'-hydroxyphenyl)-trans-but-3-en-2-one (7) inhibited Pseudomonas syringae and Bacillus sphaericus. It is therefore evident that all the isolates of Piper longum had antibacterial activity except 3-(3'-4'-5'-trimethoxyphenyl) propionicacid (4), whereas isolates of T. baccata showed moderate activity.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.

Striking differences in the production of specific inhibitory agents affecting other strains of the same (or of related) species were found between genera of the family Enterobacteriaceae. We tested 50-163 strains each of the potentially pathogenic genera: Escherichia, Citrobacter, Enterobacter, Kluyvera, and Leclercia for their ability to produce bacteriophages, high-molecular-weight (HMW) and low-molecular-weight (LMW) bacteriocins and siderophores against the same sets of strains, using the cross-test method. The genus Escherichia differs substantially from all other Enterobacteriaceae, harboring a notable proportion of lysogenic (36.6%) and colicinogenic (13.9%) strains. Only 18.2% of the Citrobacter strains are lysogenic and only rarely are they colicinogenic, although in 7.3%, they produce phage tail-like bacteriocins. On the other hand, Kluyvera strains were only in 1.8% lysogenic, no colicinogenic strains were found, but in 7.3%, they produced siderophores causing zones of growth inhibition in agar cultures of strains of the same genus. In Leclercia, 10.0% of the strains were lysogenic, 2.0% produced HMW bacteriocins, no colicinogenic strains were found and 2.0% produced siderophores. Enterobacter has shown 23.1% of strains producing siderophores, whereas merely 7.7% were lysogenic, 1.9% colicinogenic and 3.8% formed phage tail-like bacteriocins. HMW bacteriocins of Enterobacter strains disposed of an unusually wide spectrum of activity. The siderophore activity spectrum was rather wide in any genus, but the siderophores were usually not produced by strains producing phages or colicins.

Differential pathogenicity of two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle, Tribolium castaneum

Simple SummaryThe tiger mosquito is a novel vector for a variety of viral diseases in Colombia. Glyphosate herbicides have been extensively used in the country as a means to battle illicit crops, namely coca. Negative effects of this compound on arthropods have been reported, but no emphasis on dipterans has been evaluated. Different bacilli bacteria, including the Colombian Lysinibacillus sphaericus, have shown mosquitocidal potential through the production of different proteins. The surface layer (S-Layer) protein, present in this bacterial species, is involved in normal processes, such as protection and shape, but it has been reported as having a role in the mosquitocidal action of the species. In this paper, we evaluate the toxicity of glyphosate, its derivates, and the bacterial S-Layer protein on tiger mosquito larvae, as well as the synergic effect these compounds may have. Bacterial-derived formulations may provide an alternative to chemical pest control and be a viable way to remediate environmental contamination consequences of the drug war.Glyphosate and glyphosate-based herbicides are among the most used chemicals in plant pest control. Both glyphosate and its main by-product Aminomethylphosphonic Acid (AMPA) are highly environmentally persistent and, through several processes (including surface runoff and bioaccumulation), affect species beyond their intended targets, especially in aquatic ecosystems. Aedes albopictus is a novel invasive arboviral vector in Colombia and has spread to much of the national territory in recent years. Strains of the bacterium Lysinibacillus sphaericus have shown the ability to degrade glyphosate into environmentally inert compounds, in addition to having great larvicidal efficiency in different mosquito species through the production of several proteins, including the surface layer (S-Layer) protein. The S-Layer is a bacterial structure consisting of glycoprotein monomers, and its functions are thought to include bacterial interactions, protection from the outside medium and biological control. The study assessed the entomopathogenic activity of L. sphaericus S-Layer protein on Ae. albopictus larvae, and the effects that glyphosate and its by-products have in this process. To that end, bioassays were performed to compare the larval mortality between different treatments with and without S-Layer, glyphosate, and glyphosate derivates. Comparisons were made through Analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) analyses. Significant differences were found in larval mortality in the treatments, and larval mortality was greater when the S-Layer protein was present, though glyphosate field-doses (1.69 g/L) alone had a notable toxicity as well. An apparent synergic effect on the mortality of larval Ae. albopictus when exposed to mixtures containing 1500 ppm of the S-Layer protein, glyphosate, and/or glyphosate derivates was found. Further studies are needed for the in-depth understanding of this mechanism and its consequences on aquatic ecosystems.

Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.

Xenorhabdus nematophila (Xn)와 Photorhabdus tempeerata subsp. temperata (Ptt)의 곤충병원세균을 배추좀나방(Plutella xylostella)의 혈강에 주입할 경우 높은 병원력을 보였다. 본 연구는 이들 세균 배양액의 섭식 처리에 따른 배추좀나방에 대한 병원성 유기를 조사하였다. 세균 배양액만을 이용하여 배추좀나방 3령충에 섭식 처리한 결과 뚜렷한 병원성을 유발하지 못하였으나, Bacillus thurigiensis(Bt) 와 혼합 처리하였을 때 높은 Bt 병원성 제고 효과를 나타냈다. 물질 추적을 위해서 이 세균 배양액을 유기 용매를 이용하여 헥산, 에틸아세테이트 및 수용액 추출 분획구로 분리하였다. 대부분이 Bt 상승효과는 에틸아세테이트 추출 분획구에서 나타났다. Thin layer chromatography 분석 결과는 에틸아세테이트 분획구가 대사물질을 포함하고 있으며, 이들이 헥산 또는 수용액 추출 분획구에 포함된 물질과는 상이하다는 것을 나타냈다. 이러한 결과는 이들 곤충병원세균이 Bt 병원성을 제고시키는 물질을 생산하고 배양액으로 분비한다고 제시하고 있다. Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.