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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses.

A mutant defective in aciduricity, GS5Tn1, was constructed following mutagenesis of Streptococcus mutans GS5 with the conjugative transposon Tn916. The mutant grew poorly at acidic pH levels and was sensitive to high osmolarity and elevated temperatures. These properties resulted from a single insertion of Tn916 into the GS5 chromosome, and the DNA fragment harboring the transposon was isolated into the cosmid vector, charomid 9-20. Spontaneous excision of Tn916 from the cosmid revealed that Tn916 inserted into a 8.6-kb EcoRI fragment. On the basis of the restriction analyses of insert fragments, it was found that Tn916 inserted into a 0.9-kb EcoRI-XbaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames, ORF1 and ORF2. By using a marker rescue strategy, a 6.0-kb HindIII fragment including the target site for Tn916 insertion and the 5' end of ORF1 was isolated and sequenced. The deduced amino acid sequences of ORF1 and ORF2 showed significant homology with the diacylglycerol kinase and Era proteins, respectively, from Escherichia coli. Nucleotide sequence analysis of the Tn916 insertion junction region in the GS5Tn1 chromosome revealed that the transposon inserted near the 3' terminus of ORF1. Restoration of ORF1 to its original sequence in mutant GS5Tn1 was carried out following transformation with integration vector pVA891 containing an intact ORF1. The resultant transformant showed wild-type levels of aciduricity as well as resistance to elevated temperatures and high osmolarity. These results suggest that the S. mutans homolog of diacylglycerol kinase is important for adaptation of the organism to several environmental stress signals.     

Physical characterization of katG, encoding catalase HPI of Escherichia coli

The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions. This fragment was subcloned into the HindIII site of pAT153 to create pBT22. The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx. 2.5 kb as determined by complementation and expression in maxicell strains. Further reduction in size or digestion from the opposite end inactivated the gene. The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8. Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI.     

Physical and functional map of an amikacin-resistance plasmid isolated from a multiresistant strain of Serratia marcescens

pTK159, a multiresistance 40-kilobase (kb) plasmid, was isolated from a clinical strain of Serratia marcescens. pTK159 was nonconjugative and carried determinants for resistance to amikacin, streptomycin/spectinomycin, sulfamethoxazole and ampicillin. A physical and functional map of pTK159 was constructed. By cloning various fragments of pTK159 in pACYC184 or pBR322, genes for resistance to amikacin, streptomycin/spectinomycin, and sulfamethoxazole were found to be located on a 2.0-kb BamHI-HindIII fragment, a 1.4-kb HindIII fragment and a 2.1-kb HindIII fragment, respectively. The map of pTK159 was compared with published maps of amikacin-resistance determinants and transposons.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

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.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.