Restriction map of the Serratia entomophila plasmid pADAP carrying virulence factors for Costelytra zealandica.

Some strains of the Enterobacteriaceae Serratia entomophila and S. proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The virulence determinants of the disease reside on a large plasmid designated pADAP (amber disease-associated plasmid). A BamHI, EcoRI, and HindIII restriction cleavage map of pADAP was constructed by means of cloning restriction fragments. Each fragment was mapped, and neighboring fragments of mapped clones were systematically isolated from libraries using DNA probes constructed from previously cloned fragments. Through the use of sniff sequencing from the distal ends of a number of pADAP subclones the location of putative IS elements and genes involved in replication and conjugation were identified and assigned on the map. The location of the amber disease virulence-associated region was also mapped. The final map of pADAP spans 155 kb, 40 kb larger than the previous estimate.     

Occurrence of sep insecticidal toxin complex genes in Serratia spp. and Yersinia frederiksenii

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. Sequence analysis suggests that the sepABC gene cluster may be part of a horizontally mobile region. This study presents evidence for the putative mobility of the sep genes of pADAP. Southern blot analysis showed that orthologues of the sep genes reside on plasmids within S. entomophila, S. liquefaciens, S. proteamaculans, and a plasmid from Yersinia frederiksenii. Three plasmids hybridized to the pADAP sep virulence-associated region but not the pADAP replication and conjugation regions. Subsequent DNA sequence analysis of the Y. frederiksenii sep-like genes, designated tcYF1 and tcYF2, showed that they had 88% and 87% DNA identity to sepA and sepB, respectively. These results indicate that the sep genes are part of a discrete horizontally mobile region.     

Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.     

Virulence of Serratia strains against Costelytra zealandica

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.     

Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica

Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues.     

Peripheral sequences of the Serratia entomophila pADAP virulence-associated region

Some strains of the Enterobacteriaceae Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The genes responsible for this disease reside on a large, 155-kb plasmid designated amber disease-associated plasmid (pADAP). Herein, we report the DNA sequencing of approximately 50 kb upstream and 10 kb downstream of the virulence-encoding region. Based on similarity with proteins in the current databases, and potential ribosome-binding sites, 63 potential ORFs were determined. Eleven of these ORFs belong to a type IV pilus cluster (pilL-V) and a further eight have similarities to the translated products of the plasmid transfer traH-N genes of the plasmid R64. In addition, a degenerate 785-nt direct repeat flanks a 44.7-kb region with the potential to encode three Bacillus subtilis Yee-type proteins, a fimbrial gene cluster, the sep virulence-associated genes and several remnant IS elements.     

Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.     

Plasmid transfer among several members of the family Enterobacteriaceae increases the number of species capable of causing experimental amber disease in grass grub

Abstract Ability to cause amber disease in the New Zealand grass grub, Costleytra zealandica (Coleoptera: Scarabaeidae), by Serratia entomophila and S. proteamaculans (Enterobacteriaceae), is dependent on the presence of a large plasmid in bacterial strains. Transfer of the plasmid alone to several other Enterobacteriaceae resulted in the ability to cause the disease in grass grub larvae. No species other than S . entomophila or S . proteamaculans has previously been recorded causing amber disease.     

Isolation and enumeration of Serratia entomophila—a bacterial pathogen of the New Zealand grass grub, Costelytra zealandica

Several agar media were tested for their use in a selective isolation and identification scheme for Serratia entomophila , a bacterium causing amber disease of the New Zealand grass grub, Costelytra zealandica (White). Soil dilutions were plated on caprylate thallous agar (CTA), selective for Serratia spp. Most strains of Ser. entomophila grew well on CTA; the mean efficiency of colony formation on CTA was 94 ± 3% of that on a non-selective medium. The identity of colonies growing on CTA was determined on the basis of their growth reactions on DNase-toluidine blue agar, adonitol agar and itaconate agar. Serratia entomophila could be distinguished from other Serratia spp. found in New Zealand soils, in particular Ser. proteamaculans , another causal agent of amber disease of grass grub. The identification scheme allowed the selective recovery of Ser. entomophila from field soils containing a diverse microflora.     

Induced expression of the Serratia entomophila Sep proteins shows activity towards the larvae of the New Zealand grass grub Costelytra zealandica

Serratia entomophila and Serratia proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. The translated products of the sep genes are members of the toxin complex (Tc) family of insecticidal toxins that reside in the genomes of some Enterobacteriaceae. Each of the sep genes was placed either singly or as various combinations under the control of an inducible arabinose promoter, allowing their inductive expression. Western Immunoblot confirmed that each of the Sep proteins migrated at their predicted size on sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. Bioassays of sonicated filtrates derived from the various arabinose-induced para-SEP constructs showed that only when sepA, sepB and sepC were coexpressed were amber disease symptoms observed in grass grub larvae. Fourteen days after ingestion of the Sep protein filtrate, approximately 64% of the larvae reverted from a diseased to a healthy phenotype. Redosing the revertents with a fresh Sep protein filtrate reinitiated the amber pathotype, indicating that the Sep proteins are needed to be continuously present to exert an effect.     

Pathobiology of amber disease,caused by Serratia Spp., in the New Zealand grass grub,Costelytra zealandica

Amber disease in the New Zealand grass grub (Costelytra zealandica) is caused by some strains of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae). When treated with pathogenic isolates, larvae ceased feeding within 48 h, developed an amber coloration after 72 h, and entered a long chronic phase without feeding. An acute dose of 2-4 x 10(4) pathogenic bacteria was sufficient to produce disease in 50% of treated larvae. Time to death was directly related to temperature. At 15 degrees C, infected larvae remained in a chronic, nonfeeding state for more than 4 months prior to death. Nonpathogenic isolates, lacking the disease-causing plasmid (pADAP), had no effect on either feeding or disease. Twenty-four hours after ingestion, bacteria were found predominantly in the hindgut and growth occurred primarily within the fermentation chamber and in the head section of the larvae. Nonpathogenic strains did not multiply in treated larvae. Treatment of diseased larvae with antibiotic eliminated Serratia cells from the insects but did not result in restoration of feeding or the dark gut characteristic of the healthy larva.     

Expression of the antifeeding gene anfA1 in Serratia entomophila requires rpoS

The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS-mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease.     

Use of the green fluorescent protein to monitor the fate of Serratia entomophila causing amber disease in the New Zealand grass grub, Costelytra zealandica

A series of constitutive green fluorescent protein (pGFPuv) derivatives of the bacterium Serratia entomophila (Enterobacteriaceae) were constructed, allowing the fate of cells causing amber disease ingested by the New Zealand grass grub (Costelytra zealandica, Coleoptera: Scarabaeidae) to be monitored. Examination of tissue and contents of the alimentary tract over time from ingestion, under fluorescence microscopy, revealed that the major site of S. entomophila colonisation in the grass grub is intestinal particulate matter. Visual examinations showed that wild type pathogenic strain persisted in high numbers in the grass grub intestinal tract, notably in the area of the hindgut, but the S. entomophila pADAP-free strain 5.6RC and the pADK mutant derivatives (pADK-4, -10, -13) that gave a non-feeding without gut clearance phenotype, were unable to colonise the gut. The indiscriminate colonisation of the intestinal tract particulate matter by pathogenic bacteria, rather than the colonisation of a specific site of activity, suggests that the bacterial toxins are induced and released from the bacteria while they live freely in the grass grub intestinal tract.     

Virulence of Serratia Strains against Costelytra zealandica

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.     

Microbes, models and management of the New Zealand grass grub,

[First paragraph]Improved grasslands pose particular problems in pest management. The areas are extensive and the returns per hectare from grassland agriculture are much less than those from intensive cropping or horticulture, but pastures are usually sown to last for a number of years and have a much more stable ecology than in cropping or horticulture. For these reasons, biological controls and plant resistance have long been the preferred options for managing pasture pests. Within this context, the role of diseases in pasture pest population dynamics has received increasing attention, especially their ability to control pest outbreaks. Diseases are common within our major pasture pest species but their role in population regulation is often difficult to define. One of New Zealand’s major pasture pest species, the grass grub Costelytra zealandica, is widespread and often damaging throughout the country. The insect is an endemic species that has adapted to an introduced pasture system, dominated by exotic plant species (Lolium perene/Trifolium repens), where it can reach populations ten-fold higher than in its native habitat. Such high densities favour disease transmission and it is not surprising that a wide array of pathogens have been recorded from this insect (Glare et al., 1993). But the insect can still be highly damaging and can cause total loss of sown species within 3–4 years from sowing in grass grub prone regions. Probably the most important of these diseases, found throughout New Zealand, is amber disease caused by strains of the bacteria Serratia entomophila and S. proteamaculans. This is an unusual disease, controlled by a bacterial plasmid which has only been found in New Zealand bacterial isolates (Jackson et al., 2001).     

Preliminary characterization of bacteriophages of Serratia entomophila

C.R. WILSON, T.A. JACKSON AND H.K. MAHANTY. 1993. Bacteriophage was found for the first time associated with the bacterium Serratia entomophila, a pathogen of the New Zealand grass grub ( Costelytra zealandica ). Phage was isolated from the homogenized guts of field-collected grass grubs from sites throughout New Zealand. The main phage type, φCW1, produced opaque plaques in sensitive bacterial lawn and had a lamboid structure consisting of an icosahedral head (55 nm) and a long non-contractile tail (175 times 17 nm) with a bar across the base of the tail. Nucleic acid from φCW1 was digested to nucleotides by DNAse, suggesting double stranded DNA. On further examination of the homogenates, five phage types, φCW1-5, could be distinguished on the basis of plaque morphology. Bacterial host range was determined by testing against a selection of Serratia spp. and other bacteria. All five phage types lysed Ser. entomophila only. Differences in susceptibility to the phage types were found within this species. Lysogeny was demonstrated in φCW1 by immunity to superinfection and induction of free bacteriophage from suspected lysogens. A restriction map for φCW1 was determined with Bam HI, Eco RI and Hind III and a postulated origin of replication (ori) and cohesive site (cos) was suggested. The possible implications of bacteriophage on the use of Ser. entomophila as a biological control agent are discussed.     

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.     

Evolution of virulence in a novel family of transmissible mega-plasmids

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; a mber d isease-a ssociated p lasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.     

Adhesion of bacteria (Serratia spp.) to the foregut of grass grub (Costelytra zealandica (white)) larvae and its relationship to the development of amber disease

Adhesion of pathogenic strains of Serratia spp. to the foregut tissue of the New Zealand grass grub (Costelytra zealandica) was shown to be associated with the development of amber disease. Bacteria were always found adhering to the crop in the region of the cardiac valve in larvae showing disease symptoms after in vivo treatment with pathogenic bacteria while no significant colonization was observed in larvae treated with wild‐type, non‐pathogenic strains. The in vitro inoculation of excised crops with pathogenic and non‐pathogenic strains resulted in a similar pattern of adhesion. It is suggested that adhesion is an early step in pathogenesis and that farther bacterial mediated factors could be required for fall expression of amber disease.