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.     

Nucleotide sequence of the Serratia entomophila plasmid pADAP and the Serratia proteamaculans pU143 plasmid virulence associated region

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The disease determinants of S. entomophila, are encoded on a 153,404-bp plasmid, termed pADAP for amber disease associated plasmid. The S. proteamaculans strain 143 (Sp143) exhibits an unusual pathotype, where only 60-70% of C. zealandica larvae infected with the bacterium succumb to disease. DNA sequence analysis of the Sp143 pU143 virulence associated region identified high DNA similarity to the pADAP sep virulence associated region, with DNA sequence variation in the sepA gene and the variable region of the sepC component. No pADAP anti-feeding prophage orthologue was detected in the Sp143 genome. The region of pADAP replication was cloned and found to replicate in S. entomophila but not in Escherichia coli. DNA sequence analysis of the plasmid pSG348 repA gene from the French isolate of Serratia grimesii, identified 93% DNA identity to the pADAP repA gene. A comparison of the pU143 virulence associated region with the completed pADAP nucleotide sequence is given.     

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.     

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.     

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.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

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.     

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.     

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.     

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.     

Quantification and kinetics of the decline in grass grub endopeptidase activity during initiation of amber disease

Amber disease in the grass grub (Costelytra zealandica White) (Coleoptera: Scarabaeidae), caused by strains of the bacteria Serratia entomophila or S. proteamaculans, is characterised by cessation of feeding and clearance of the midgut. Analysis of the midgut enzyme activity in diseased grass grub larvae showed that proteolytic activity was reduced to low levels. The endopeptidases, trypsin, elastase, and chymotrypsin, were all markedly reduced in activity whereas the exopeptidases (leucine-aminopeptidase and carboxypeptidase A and B) were much less affected. There was no effect on the non-proteolytic enzymes, esterase and alpha-amylase. Sequential analysis of enzyme levels in the gut during onset of disease showed that proteolytic activity dropped after cessation of feeding and preceded gut clearance. In starved, uninfected larvae enzyme activity levels remained high, indicating that decline in enzyme activity is not associated with absence of food and cessation of feeding, but with the onset of 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.     

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.     

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.     

Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.     

The lipopolysaccharide biosynthesis core of the Mexican pathogenic strain Serratia entomophila is associated with toxicity to larvae of Phyllophaga blanchardi

The Mexican bacteria Serratia entomophila strain Mor4.1 (Enterobacteriaceae) is pathogenic to coleopteran species of the Phyllophaga genus, which are considered important soil-dwelling pests. Mor4.1 causes anti-feeding activity and mortality to larvae after oral and injection bio-assay either, by bacteria or by cell free culture broth inoculation. The pathogenic determinants of Mor4.1 have not been elucidated. We hypothesize that Mor4.1 produces several toxins and other virulence factors, some acting at the level of the insect gut and others at the hemocoel. To identify and characterize virulence factors, a fosmid library of S. entomophila Mor4.1 was made in Escherichia coli. Five different insecticidal clones were isolated by injecting individual clones into Phyllophaga blanchardi larvae. The complete 40 kb DNA sequence and gene organization of clone G8 was determined. By comparative genomics, 21 genes were associated with virulence. By transposon (Tn5) insertion mutagenesis of G8 and further bio-assays we show that a dUTPase, a flavoprotein and a heptosyltransferase III, are key factors for G8 toxic activity. The heptosyltransferase III, is part of the lipopolysaccharide (LPS) biosynthesis core. We demonstrated that purified LPS from G8 and Mor4.1 are toxic to P. blanchardi larvae by injection bio-assay.     

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.     

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.