An amoeba phagocytosis model reveals a novel developmental switch in the insect pathogen Bacillus thuringiensis.

The Bacillus cereus group bacteria contain pathogens of economic and medical importance. From security and health perspectives, the lethal mammalian pathogen Bacillus anthracis remains a serious threat. In addition the potent insect pathogen Bacillus thuringiensis is extensively used as a biological control agent for insect pests. This relies upon the industrial scale induction of bacterial spore formation with the associated production of orally toxic Cry-toxins. Understanding the ecology and potential alternative developmental fates of these bacteria is therefore important. Here we describe the use of an amoeba host model to investigate the influence of environmental bactivorous protists on both spores and vegetative cells of these pathogens. We demonstrate that the bacteria can respond to different densities of amoeba by adopting different behaviours and developmental fates. We show that spores will germinate in response to factors excreted by the amoeba, and that the bacteria can grow and reproduce on these factors. We show that in low densities of amoeba, that the bacteria will seek to colonise the surface of the amoeba as micro-colonies, resisting phagocytosis. At high amoeba densities, the bacteria change morphology into long filaments and macroscopic rope-like structures which cannot be ingested due to size exclusion. We suggest these developmental fates are likely to be important both in the ecology of these bacteria and also during animal host colonisation and immune evasion.

Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae): the hidden host of Photorhabdus asymbiotica (Enterobacteriaceae: gamma-Proteobacteria).

A new entomopathogenic nematode species from Australia, Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae) is described. Morphological and molecular studies together with cross-hybridization tests indicated that this nematode represents a new undescribed species, closely related to members in the 'indica-group'. However, the new species can be distinguished from other species in this genus by a combination of several qualitative and quantitative morphological traits. Key diagnostic features include: body size and excretory pore position of the third-stage infective juveniles; male bursa with a reduction of bursal rays, usually affecting the terminal set of papillae, with symmetrical or asymmetrical loss of one or two pairs; vulva of hermaphrodites more anteriorly located than in other species in the indica-group (V% average: 43), with non-protruding or slightly protruding lips, and longer tail length (average: 106 mum). The new species can be further characterized by molecular traits of sequence data from the internal transcribed spacer (ITS) region of ribosomal DNA. Additionally, the bacterial symbiont of this new species, Photorhabdus asymbiotica Kingscliff strain, was phenotypically characterized and compared with other P. asymbiotica strains. The Kingscliff strain revealed many characters not present in other strains of this species. We hypothesize that the newly found traits may contribute to the maintenance of this mutualistic association of the bacterium with its nematode host.

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.

Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.

Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria.

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.

The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells.

Photorhabdus bacteria produce a number of toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy (Mcf), is sufficient to allow Escherichia coli to persist within and kill caterpillars. Mcf causes shedding of the insect midgut epithelium and destructive blebbing of haemocytes suggesting it may trigger apoptosis. To investigate this hypothesis, here we examine the effects of E. coli-expressed Mcf on the mammalian cell lines COS-7, NIH 3T3 and HeLa cells. Cells treated with Mcf show apoptotic nuclear morphology, active caspase-3, DNA laddering after 6 h, and the presence of cleaved PARP after 16 h. These effects are prevented by the apoptosis inhibitor zVAD-fmk. Transfection of cells with constructs expressing only the NH2-terminal 1280 amino acids of Mcf, as a fusion with Myc, also triggered cell destruction. The expressed fusion protein was concentrated into the Golgi apparatus before cell death. These results confirm that the novel insecticidal toxin Mcf induces apoptosis but the precise intracellular pathway remains obscure.

A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects.

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.

Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sexta when expression in E. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. luminescens K122 and E. coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli produces visible approximately 25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.

Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection.

During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.

The tc genes of Photorhabdus: a growing family.

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.

A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.

Transcription analysis of the prolate-headed lactococcal bacteriophage c2.

A detailed transcription map of the prolate-headed lactococcal phage c2 has been constructed. Transcription of about one-third of the genome, encoding 22 open reading frames, began within the first 2 min of infection and produced at least 12 overlapping transcripts that persisted until lysis occurred at 30 min after initiation of infection. The remaining two-thirds of the genome, encoding 17 open reading frames, was divergently transcribed, beginning between 4 and 6 min after initiation of infection, and resulted in at least 18 overlapping transcripts that persisted until lysis. Five very strong, simultaneously active, and probably unregulated early promoters and a single positively regulated late promoter were identified. The late promoter had an extended -10 sequence, had a significant basal level of activity in the uninduced state, and was induced to high activity by a phage gene product. The complex overlapping pattern of transcripts resulted from the action of the multiple early promoters, inefficient termination of transcription, and (possibly) processing of a late precursor transcript(s). Phage proteins were not required for these processes, and the host RNA polymerase was probably used for both early and late transcription.

An origin of DNA replication from Lactococcus lactis bacteriophage c2.

An origin of DNA relication was identified in the intergenic region between the early and late gene regions of prolate lactococcal phage c2. A DNA fragment containing this origin, designated ori, was shown to direct DNA replication in Lactococcus lactis but not in Escherichia coli. A comparison of ori with the corresponding regions of other prolate phages revealed strict conservation of the nucleotide sequence in one half of this intergenic region. This conserved region alone would not support DNA replication. No open reading frames were identified in the ori fragment, suggesting that host factors alone are sufficient to initiate DNA replication at ori. A novel class of lactococcal vectors and E. coli-L. lactis shuttle vectors based on ori have been constructed.

Sequencing and analysis of the prolate-headed lactococcal bacteriophage c2 genome and identification of the structural genes.

The 22,163-bp genome of the lactococcal prolate-headed phage c2 was sequenced. Thirty-nine open reading frames (ORFs), early and late promoters, and a putative transcription terminator were identified. Twenty-two ORFs were in the early gene region, and 17 were in the late gene region. Putative genes for a DNA polymerase, a recombination protein, a sigma factor protein, a transcription regulatory protein, holin proteins, and a terminase were identified. Transcription of the early and late genes proceeded divergently from a noncoding 611-bp region. A 521-bp fragment contained within the 611-bp intergenic region could act as an origin of replication in Lactococcus lactis. Three major structural proteins, with sizes of 175, 90, and 29 kDa, and eight minor proteins, with sizes of 143, 82, 66, 60, 44, 42, 32, and 28 kDa, were identified. Several of these proteins appeared to be posttranslationally modified by proteolytic cleavage. The 175- and 90-kDa proteins were identified as the major phage head proteins, and the 29- and 60-kDa proteins were identified as the major tail protein and (possibly) the tail adsorption protein, respectively. The head proteins appeared to be covalently linked multimers of the same 30-kDa gene product. Phage c2 and prolate-headed lactococcal phage bIL67 (C. Schouler, S. D. Ehrlich, and M.-C. Chopin, Microbiology 140:3061-3069, 1994) shared 80% nucleotide sequence identity. However, several DNA deletions or insertions which corresponded to the loss or acquisition of specific ORFs, respectively, were noted. The identification of direct nucleotide repeats flanking these sequences indicated that recombination may be important in the evolution of these phages.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis.

18 different promoter elements, encompassing a 71-fold range of activity, were isolated from the chromosome of Lactococcus lactis (Ll) MG1363 and from an uncharacterised small isometric bacteriophage of Ll. The Vibrio fischeri (Vf) luciferase-encoding gene (lux) was used as a reporter in Ll, so that the promoters could be identified strictly on the basis of their activity in the homologous host. Sequence and primer extension analysis of six of the promoters has provided a new consensus sequence for the -35 and -10 hexanucleotide motifs present upstream from lactococcal transcription start points. When the nucleotide sequence of the most active promoter (P15) was compared with that of the highly expressed Ll usp45 gene, a novel 8-bp region of homology was identified which corresponded to the newly derived consensus -35 sequence element; this element may therefore be of general importance in Ll gene expression. The isolation of these promoters has also enabled us to investigate the characteristics of the Vf Lux activity in Ll under different physiological conditions using promoters of different strengths. Lux activity in Ll is critically dependent upon the phase of cell growth. Luminescence falls sharply in stationary phase, possibly due to a lack of FMNH2. In contrast to the kinetics of Lux function in Escherichia coli (Ec), Lux activity in Ll declines rapidly after addition of the substrate; the rate of decay is dependent both on the growth phase and on the strength of the promoter. It is apparent that the previously reported thermal instability of Lux is in fact a function of the host organism in which Lux is expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular biology of lactococcal bacteriophage c2.

The 22163 bp genome of the lytic prolate-headed lactococcal phage c2 was fully sequenced. Mapping of restriction sites and RNA transcripts demonstrated the presence of early and late genes. Early and late promoters were identified. The early region contained 21 ORFs, with predicted protein products of 4.4-32.9 kDA, all reading right to left. Significant similarity was found between a putative protein encoded by an early region ORF and the erf (essential recombination function) gene product of Salmonella phage P22. The late genes for which a function has been identified, all of which read from left to right, included a possible holin gene, and genes encoding three major and six minor phage structural proteins. Analysis of the cohesive termini revealed complementary, non-symmetrical, 9-base single-stranded 3' extended DNAs. The exploitation of phage sequence data and analysis of phage genomes to find ways of inhibiting phage replication is discussed.