Bacillus Popilliae Research Articles

Paenibacillus konsidensis sp. nov., isolated from a patient

A Gram-positive, rod-shaped bacterium, designated strain LBY(T), was isolated from the blood of a Korean patient. The organism could not be identified based on conventional clinical microbiological methods. 16S rRNA gene sequencing and phylogenetic analysis revealed that it belonged to the genus Paenibacillus, but was distinct from recognized Paenibacillus species. Strain LBY(T) was related most closely to the type strains of Paenibacillus macerans and Paenibacillus popilliae, with 16S rRNA gene sequence similarities of 96.2 and 95.4%, respectively. The major fatty acids of strain LBY(T) were anteiso-C(15:0) (43.8%), C(16:0) (10.4%), iso-C(16:0) (10.3%), iso-C(15:0) (9.2%) and anteiso-C(17:0) (8.3%). On the basis of biochemical characteristics, fatty acid composition and comparative 16S rRNA gene sequence analysis, strain LBY(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus konsidensis sp. nov. is proposed. The type strain is LBY(T) (=ABB-ID-KSY9(T)=KCTC 13165(T)=JCM 14798(T)).

Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny

A phylogenetic grouping of 48 different isolates of milky disease bacteria isolated in the United States was determined using genomic RFLP analysis and 16S rDNA sequence comparison. A clear distinction between Paenibacillus popilliae isolates and Paenibacillus lentimorbus isolates was evident from the results of each procedure. The P. popilliae isolates segregated into two phylogenetic groups and the P. lentimorbus isolates segregated into three phylogenetic groups. In the United States, P. popilliae group 1 was generally isolated from insects collected west of the Appalachian Mountains. P. popilliae group 2 was only isolated from insects collected east of the Appalachian Mountains. P. lentimorbus groups 1 and 2 were obtained from insects collected west and south of the Appalachians. P. lentimorbus group 3 was identified in insects collected east of the mountains. From five different locations in Connecticut, 12 milky disease bacterial isolates were classified as P. popilliae and three were classified as P. lentimorbus. Except for one isolate, all P. popilliae isolates were of phylogenetic group 2. The three P. lentimorbus strains were isolated from diseased insects that had been collected from a localized area in the state. These three strains formed a separate phylogenetic grouping (i.e., group 3) of P. lentimorbus and, based on 16S rDNA sequence comparisons, were most similar to the newly identified P. lentimorbus Semadara strain recently isolated in Japan. All milky disease bacteria that had been isolated from commercially available insecticide preparations were identified as P. popilliae group 1.

A novel bacterial disease of the European shore crab, Carcinus maenas – molecular pathology and epidemiology

Several rickettsia-like diseases have been reported in arthropods (insects and crustaceans), some of which result in significant losses of economically important species such as shrimp and crabs. This study reports on the molecular pathology of a recently emerged disease of the European shore crab, Carcinus maenas, termed milky disease - named as a result of the unusual milky appearance of the haemolymph (blood). This disease was more prevalent (>26 %) during summer months when the water temperature in a pilot crab farm was approximately 19 degrees C. The putative causative agent of the disease was a Gram-negative bacterium that could not be cultured on a range of agar-based growth media. Diseased crabs showed significant reductions in free blood cell numbers and total serum protein. Such animals also displayed raised levels of glucose and ammonium in blood. Ultrastructural and in situ hybridization studies revealed that the causative agent associated with milky disease multiplied in the fixed phagocytes of the hepatopancreas (digestive gland), ultimately to be released into the haemolymph, where the circulating blood cells showed little response to the presence of these agents. Attempts to induce the infection by short-term temperature stress failed, as did transmission experiments where healthy crabs were fed infected tissues from milky disease affected individuals. Sequence analysis of the 16S rRNA gene from the milky disease bacteria indicated that they are a previously undescribed species of alpha-proteobacteria with little phylogenetic similarity to members of the order Rickettsiales.

A marine killer yeast against the pathogenic yeast strain in crab ( Portunus trituberculatus) and an optimization of the toxin production

A pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus was identified to be Metschnikowia bicuspidate according to the results of routine yeast identification and 18S rDNA and ITS sequences. After screening of more than 300 yeast strains from different sources in marine environments, it was found that strain YF07b had the highest ability to produce killer toxin against the pathogenic yeast. Strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The optimal conditions for killer toxin production by strain YF07b were the production medium with 2.0% NaCl, pH 4.5, cultivation temperature of 20 degrees C and the optimal conditions for action of the crude killer toxin against the pathogenic yeast were the assay medium with 6.0% NaCl, pH 4.5 and temperature 15 degrees C.

Putative VanRS-Like Two-Component Regulatory System Associated with the Inducible Glycopeptide Resistance Cluster of Paenibacillus popilliae

Paenibacillus popilliae contains vanF encoding a putative D-Ala:D-lactate (D-Lac) ligase, VanF, as part of the vanY(F)Z(F)H(F)FX(F) cluster that is similar in structure to the enterococcal vanA and vanB clusters. Using growth curves, we demonstrated that vancomycin resistance in P. popilliae is inducible. Using degenerate oligonucleotides targeted at bacterial cell wall ligases, we identified a second ligase gene with features of a D-Ala:D-Ala ligase in both P. popilliae and the related, vancomycin-susceptible, Paenibacillus lentimorbus. The 3,380-bp region upstream of vanY(F)Z(F)H(F)FX(F) in P. popilliae ATCC 14706 was sequenced and found to contain genes encoding a putative two-component regulator, VanR(F)S(F), similar to VanRS but more closely related to a family of two-component regulators linked to VanY-like carboxypeptidases in several glycopeptide-susceptible Bacillus species. This upstream region also included a transposase similar to a transposase found in Bacillus halodurans and, in some strains, a 99-bp insertion of unknown function with 95% nucleotide identity to a portion of the Tn1546 transposase gene. Analysis of glycopeptide resistance-associated clusters from soil and/or insect-dwelling organisms may provide important clues to the molecular evolution of acquired glycopeptide resistance elements in human pathogens.

Characterization ofPaenibacillus popilliaerRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.

A new strain of Paenibacillus lentimorbus isolated from larvae of the oriental beetle, Blitopertha orientalis (Coleoptera: Scarabaeidae), in Chiba Prefecture, Japan

In the summer of 1997, larvae of the oriental beetle, Blitopertha orientalis, with milky disease were collected from a turf field in Chiba Prefecture, Japan. Characteristic footprint-shaped sporangia containing a spore and a parasporal body were isolated from the larval hemolymph. Examination of the bacteriological characteristics of this strain revealed that it was unable to grow in 2% NaCl, was susceptible to vancomycin, and had two plasmids, of about 9.8 and 5.5 kb. The sequence of the 16S ribosomal RNA gene (16S rDNA) of this strain was more homologous with that of Paenibacillus (Bacillus) lentimorbus ATCC 14707T than those of the most famous, traditional American culture, Paenibacillus (Bacillus) popilliae ATCC 14706T and NRRL B-4081. From homology of the 16S rDNA, this strain was identified as P. lentimorbus, and named strain ‘Semadara’. The sequence of strain Semadara was highly homologous with those of three Japanese isolates of B. popilliae. Strain Semadara also infected four other scarabaeids: Anomala cuprea, Anomala rufocuprea, Phyllopertha diversa, and Popillia japonica. When second or third stadium larvae of each of the four scarabaeids were inoculated with sporangia of strain Semadara at a concentration of 1.5×108 sporangia/cup, most of the larvae died 2–4 weeks after inoculation.

Lack of interaction between the milky disease bacterium Paenibacillus popilliae and stilbene-derived optical brighteners in Japanese beetle larvae

Optical brighteners cansynergistically enhance nucleopolyhedrovirusinfectivity to lepidopteran larvae by blockingthe sloughing of infected primary midgut cellsand inhibiting the formation of the peritrophicmembrane in the hosts. Because of similaritiesin the route of infection, we investigatedwhether optical brighteners would also enhanceinfection with the milky disease bacterium,Paenibacillus popilliae, of Japanesebeetle, Popillia japonica, larvae. Thelarvae were kept in soil mixed with P.popilliae spore preparations and the opticalbrighteners Blankophor BBH, P167, or RKH withperennial ryegrass provided as food. Noenhancing effect of any of the opticalbrighteners on P. popilliae infection wasobserved at a concentration of 0.1% (w/w). Rather, when mixed into the soil at 0.02, 0.1,or 0.5% (w/w) BBH reduced P. popilliaeinfection at the highest rate.

Pulsed-field gel electrophoresis for the identification of bacteria causing milky disease in scarab larvae.

Pulsed-field gel electrophoresis for the identification of bacteria causing milky disease in scarab larvae.

Insecticidal Activity of Bacillus popilliae strain Semadara to Scarab Beetle Larvae

Insecticidal Activity of <I>Bacillus popilliae</I> strain Semadara to Scarab Beetle Larvae

Absence of Interaction between Endophytic Perennial Ryegrass and Susceptibility of Japanese Beetle (Coleoptera: Scarabaeidae) Grubs to Paenibacillus popilliae Dutky1

Absence of Interaction between Endophytic Perennial Ryegrass and Susceptibility of Japanese Beetle (Coleoptera: Scarabaeidae) Grubs to Paenibacillus popilliae Dutky1

Paenibacillus Associated with Milky Disease in Central and South American Scarabs

Thirty-one isolates of bacteria causing milky disease in scarab larvae collected in Central and South America were identified as Paenibacillus popilliae or Paenibacillus lentimorbus by use of DNA similarity analysis. The isolates were more similar to each other than to the North American isolates that are the type strains of the species. All of the bacteria of both species produced parasporal bodies, a characteristic previously believed to be unique to P. popilliae. Screening of the bacteria using PCR with parasporal protein primers revealed differences among the parasporal protein genes of P. popilliae isolates and between the parasporal genes of P. popilliae and P. lentimorbus. In contrast to P. popilliae from North America, none of the isolates from Central and South America was resistant to vancomycin, an indication of an interesting geographic distribution of the resistance genes.

Enterococcal-type glycopeptide resistance genes in non-enterococcal organisms.

Although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. The vancomycin resistance-associated genes in Enterococcus gallinarum, Enterococcus casseliflavus/flavescens, Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Erysipelothrix rhusiopathiae, are not the source of the high-level vancomycin resistance-associated genes in enterococci. There are, however, environmental organisms which have been found to have gene clusters homologous to the enterococcal vanA, vanB and vanC gene clusters; these include the biopesticide Paenibacillus popilliae, and, to a lesser extent, the glycopeptide-producing organisms Amycolatopsis orientalis and Streptomyces toyocaensis. Still, the exact sources of the enterococcal vancomycin resistance genes remain a mystery.

Enterococcal-type glycopeptide resistance genes in non-enterococcal organisms

Although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. The vancomycin resistance-associated genes in Enterococcus gallinarum, Enterococcus casseliflavus/flavescens, Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Erysipelothrix rhusiopathiae, are not the source of the high-level vancomycin resistance-associated genes in enterococci. There are, however, environmental organisms which have been found to have gene clusters homologous to the enterococcal vanA, vanB and vanC gene clusters; these include the biopesticide Paenibacillus popilliae, and, to a lesser extent, the glycopeptide-producing organisms Amycolatopsis orientalis and Streptomyces toyocaensis. Still, the exact sources of the enterococcal vancomycin resistance genes remain a mystery.

Thermobacillus xylanilyticus gen. nov., sp. nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil.

An aerobic, thermophilic, xylanolytic, spore-forming bacterium, XETP (T = type strain; P = patent strain), has been isolated from farm soil situated underneath a manure heap in northern France. Strain XETP, which stained negative in the Gram test, occurs as short rods which sometimes form chains. Its spores are ellipsoidal, central to subterminal and occur in swollen sporangia. It grows at temperatures up to 63 degrees C and in the pH range 6.5-8.5. When grown on glucose in optimal conditions, its doubling time was found to be 33 min. CO2 was observed to have a growth-stimulating effect at the start of the culture. In addition to glucose, the isolate utilizes xylose, arabinose, mannose, cellobiose, galactose, maltose, sucrose, xylan and starch. Growth is inhibited by 5% NaCl. The G+C content of strain XETP is 57.5 mol%. The 16S rDNA sequence analysis indicated that strain XETP falls into the radiation of the Bacillus-Lactobacillus-Streptococcus subdivision of the Gram-positive phylum. Its three closest phylogenetic relatives are 'Bacillus viscosus', Paenibacillus curdlanolyticus and Bacillus popilliae with identity values of 91.15, 90.94 and 90.92%, respectively. The major cellular fatty acids are 14-methyl pentadecanoic acid (16:0 iso), hexadecanoic acid (16:0) and 14-methyl hexadecanoic acid (17:0 anteiso). On the basis of 16S rRNA sequence and chemotaxonomic characteristics, the isolate is different enough for it to be considered as a member of a new genus. It is therefore proposed that this isolate represents a new genus and species: Thermobacillus xylanilyticus. Strain XETP, the type strain of Thermobacillus xylanilyticus, has been deposited in the Collection Nationale de Cultures Microbiennes (CNCM I-1017) as a patent strain.

Conjugative transposition of Tn916 and Tn925 in Bacillus popilliae

Interspecies transfer of the conjugative transposons Tn916 and Tn925 into B. popilliae Pj1 occurred using Enterococcus faecalis and Bacillus subtilis CU4049 as transposon donors. Tn916 was stably maintained in B. popilliae Pj1 following growth without selective pressure and was successfully introduced into the plasmid-containing B. popilliae strains NRRL B-2524, Ch1, and KLN4 using E. faecalis CG110. In B. popilliae, expression of the tetracycline resistant determinants on Tn916 and Tn925 provided resistance to 25 μg/mL and 50 μg/mL tetracycline, respectively. An erythromycin resistant determinant, present in Tn916ΔE, was also functional in B. popilliae Pj1 and provided resistance to 1 mg/mL erythromycin. Transfer of Tn916 into E. faecalis, B. subtilis, and between B. popilliae strains was accomplished using a transposon-containing strain of B. popilliae as donor. Efforts to transfer Tn916 between E. coli and B. popilliae were unsuccessful. Key words: Bacillus popilliae, milky disease, Tn916, conjugative transposon.

Impact of Golf Course Mowing Practices onAtaenius spretulus(Coleoptera: Scarabaeidae) and its Natural Enemies

In April of 1996 the rough-fairway interface of a golf course consisting entirely of perennial ryegrass, Lolium perenne L., was altered by implementing a new mowing regime. The experiment contained 4 replications of 4 treatments: turf mowed to fairway height 1995-1997, turf mowed to rough height 1995-1997, turf that was fairway height in 1995 and was allowed to grow to rough height in 1996-1997, and turf that was rough height in 1995 and was cut to fairway height in 1996-1997. Glass vials were used as pitfall traps to monitor activity of surface-dwelling predators and adult Ataenius spretulus (Haldeman). In 1996, the most A. spretulus larvae were found in plots where the turf was mowed to fairway height the previous year, regardless of how the turf was mowed that year. However, adult Philonthus spp. (Staphylinidae) were found in the highest numbers in the plots that were mowed to rough height, regardless of the turf height in past years. In 1997, the highest populations of A. spretulus adults and larvae were found in plots mowed to fairway height in 1996 and in 1997, regardless of 1995 mowing height. Also, twice as many larvae were infected with a milky disease causing pathogen, Bacillus sp., in the rough (68%) compared with the fairway (34%). The most prevalent staphylinids, Philonthus cognatus Stephens and P. carbonarius (Gravenhorst), were 3 times more abundant in rough-mowed turf compared with fairway-mowed turf. With grass species, irrigation, and previous pesticide use controlled by our experimental design, it appears that mowing practices are an important factor contributing to the skewed distribution of A. spretulus toward golf course fairways and away from the rough.

Susceptibility of the Taro Beetle,Papuana uninodis(Coleoptera, Scarabaeidae) to Two NewBacillus popilliaeIsolates fromPapuanaspp.

Two morphological types ofBacillus popilliae,causal agent of the milky disease, have been isolated from taro beetles (Papuanaspp, Coleoptera: Scarabaeidae).B. popilliaefromP. woodlarkiana woodlarkiana(Papua New Guinea) was a type A1 with a small sporangium (4.1×1.6 μm) and a large spore (2.1×1.4 μm) and parasporal body (1.8×1.2 μm) that sometimes overlap.B. popilliaefromP. uninodisandP. woodlarkiana laevipennis(Solomon Islands) was a type B2 with a small sporangium (2.8×1.3 μm), a small eccentric spore (1.1×0.7 μm), and no parasporal body. The infectivity of theseB. popilliaetoPapuana uninodislarvae was compared with twoB. popilliaesamples fromPopillia japonicain injection tests. The hemolymph ofP. uninodissupported the germination and growth of isolates fromPapuanaandP. japonica.Results were similar in third instars and adults. Highest infection (spores present) and mortality was caused by the isolates fromPapuana:mortality reached almost 100% 4 weeks after injection of the B2 typeB. popilliaewith 40% of larvae and 52% of adults infected. Injection of type A1 caused lower mortality but a similar percentage infected. Of two A1B. popilliaefromP. japonica,one caused a mortality comparable to type A1 fromPapuanabut lower infection; an older isolate resulted in low mortality and only one infected larva.B. popilliaetype A1 fromP. woodlarkianawas produced in the Solomon Islands by injection of spores inP. uninodis.Thirty four percent of the injected larvae and 31% of the adults produced spores with an average yield of 3.2 and 0.8 × 109spores/insect, respectively. Oral application of a single dose of 107spores of theB. popilliaeisolates fromP. uninodisorP. japonicadid not cause infection and similarly inoculation of the food with spores ofB. popilliaetype B2 did not result in infections. However, when different rates were applied to the food of second- and third-instarP. uninodis,theB. popilliaetype A1 fromP. woodlarkianacaused up to 15% infection and concentration-related mortality.

Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

Almost complete 16S rRNA gene sequences were generated for the type strains of the obligate insect pathogens Bacillus lentimorbus and Bacillus popilliae and a second strain of Bacillus popilliae (NRRL B-4081) received as 'Bacillus popilliae var. melolonthae'. A phylogenetic tree was constructed which grouped these strains into a well defined subcluster within the genus Paenibacillus. Bacillus popilliae NRRL B-4081 occupied an intermediate position between the type strains of Bacillus lentimorbus and Bacillus popilliae but with a marked clustering to the latter. The phylogenetic assignment of these strains to Paenibacillus is in contrast to earlier studies which placed these bacteria in the genus Bacillus, close to Bacillus subtilis. Indeed, the rRNA sequences generated in this study share less than 88% similarity to the deposited sequences for Bacillus popilliae ATCC 14706T and Bacillus lentimorbus ATCC 14707T. The results obtained by using different tree algorithms, bootstrap analysis, branch lengths and verification by signature nucleotide analysis supported the reclassification of these species in the genus Paenibacillus as Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

Bacillus popilliae and Bacillus lentimorbus, bacteria causing milky disease in Japanese beetles and related scarab larvae

Bacillus popilliae and Bacillus lentimorbus, causative agents of milky disease in Japanese beetle and related scarab larvae, have hitherto been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. In this study 34 isolates of these bacteria were examined for DNA similarity and by random amplified polymorphic DNA (RAPD) analysis. Two distinct but related similarity groups were identified: the first contained strains of B. popilliae and the second contained strains of B. lentimorbus. Two strains distinct from but related to B. popilliae may represent a subspecies. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae. RAPD analysis confirmed the DNA similarity results. Paraspore formation, previously believed to be a characteristic unique to B. popilliae, was found to occur among a sub-group of B. lentimorbus strains. Growth in media supplemented with 2% NaCI was found to be a somewhat less reliable characteristic in distinguishing the species than vancomycin resistance, the latter being present only in B. popilliae.