Phenotypic persistence and external shielding ultraviolet radiation inactivation kinetic model.

AIM: To develop an inactivation kinetic model to describe ultraviolet (UV) dose-response behaviour for micro-organisms that exhibit tailing using two commonly referenced causes for tailing: physical shielding of micro-organisms and phenotypic persistence. METHODS AND RESULTS: Dose-response data for Escherichia coli, Mycobacterium terrae and Bacillus subtilis spores exposed to UV radiation were fit to the phenotypic persistence and external shielding (PPES) model. The fraction of persistent micro-organisms in the original population (N(persistent)/N(total)) that exhibited reduced sensitivity to UV radiation was estimated by the PPES model as approx. 10(-7), 10(-5) and 10(-4) for E. coli, B. subtilis spores and Myco. terrae, respectively. Particle shielding effects were evaluated for Myco. terrae and resulted in additional reduction in UV sensitivity. CONCLUSIONS: Tailing occurred in laboratory experiments even when clumping and shielding were eliminated as major factors in UV resistance, suggesting that phenotypic persistence in addition to shielding may be important to consider when evaluating dose-response curves for disinfection applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The PPES model provides a mechanistically plausible tool for estimating the dose-response behaviour for micro-organisms that exhibit tailing in dispersed and aggregated settings. Accurate dose-response behaviour (including the tailing region) is critical to the analysis and validation of all UV disinfection systems.

Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation.

A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot alpha, was found only in the Bacillus cereus group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cot alpha, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a sigma(K)-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cot alpha resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot alpha gene into the mutant. Since Cot alpha is an abundant outer spore coat protein of the B. cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B. anthracis spores.

Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action.

The spore-forming bacterium Bacillus thuringiensis produces intracellular inclusions comprised of protoxins active on several orders of insects. These highly effective and specific toxins have great potential in agriculture and for the control of disease-related insect vectors. Inclusions ingested by larvae are solubilized and converted to active toxins in the midgut. There are two major classes, the cytolytic toxins and the delta-endotoxins. The former are produced by B. thuringiensis subspecies active on Diptera. The latter, which will be the focus of this review, are more prevalent and active on at least three orders of insects. They have a three-domain structure with extensive functional interactions among the domains. The initial reversible binding to receptors on larval midgut cells is largely dependent upon domains II and III. Subsequent steps involve toxin insertion into the membrane and aggregation, leading to the formation of gated, cation-selective channels. The channels are comprised of certain amphipathic helices in domain I, but the three processes of insertion, aggregation and the formation of functional channels are probably dependent upon all three domains. Lethality is believed to be due to destruction of the transmembrane potential, with the subsequent osmotic lysis of cells lining the midgut. In this review, the mode of action of these delta-endotoxins will be discussed with emphasis on unique features.

Analysis of mutations in the pore-forming region essential for insecticidal activity of a Bacillus thuringiensis delta-endotoxin.

The Bacillus thuringiensis insecticidal delta-endotoxins have a three-domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the B. thuringiensis cry1Ac1 gene, encoding parts of helix alpha4 and the loop connecting helices alpha4 and alpha5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site-directed mutations were also created in specific surface residues of helix alpha3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha4, 5 resulted in the total loss of toxicity for Manduca sexta and Heliothis virescens, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices alpha4 and alpha5 did not affect toxicity, nor did mutations in alpha3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha5, those in helix alpha4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix alpha5 is important for oligomerization and perhaps has other functions, whereas helix alpha4 must have a more direct role in establishing the properties of the channel.

Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.

Substitution of residues on the proximal side of Cry1A Bacillus thuringiensis delta-endotoxins affects irreversible binding to Manduca sexta midgut membrane.

Substitution of a positively charged residue (R93F) or addition of a negatively charged residue (A92D) at the N-terminal of alpha 3 helix of domain I of the Cry1Ac delta-endotoxin resulted in a substantial reduction in toxicity against Manduca sexta. The N-terminal residues of helix 3 are considered to be on the same (proximal) surface of the toxin as the loops in domain II which are involved in the binding of the toxin to the receptor. The loss of toxicity was not caused by a decrease in the initial binding but rather by reduced irreversible binding. Only 65 and 75% of the A92D and R93F mutant toxin, respectively, bound to midgut vesicles irreversibly, compared to 94% of the wild type toxin. On the other hand, replacing A119 in a loop on the distal side of the helices with negatively charged residues (A119D or A119E) did not affect toxicity or irreversible binding.

Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene.

Two different 30-nucleotide regions of the cryIAc insecticidal protoxin gene from Bacillus thuringiensis were randomly mutagenized. One region was within one of seven amphipathic helices believed to be important for the formation of ion channels. There was no loss of toxicity for three test insects by any of 27 mutants, a result similar to that obtained previously for mutations within another such helix. Only mutations within a region encoding the central helix have resulted in a substantial number of mutants with low or no toxicity. A second mutagenized region encodes amino acids which are unique to this toxin and are within one of the loops in a portion of the toxin important for specificity. Among 21 different mutations of these 10 residues, only changes of two adjacent serine residues resulted in decreased toxicity which was greater for Manduca sexta than for Heliothis virescens larvae. These mutant toxins bound poorly to the single M. sexta CryIAc vesicle-binding protein and to several of the multiple H. virescens-binding proteins. The loop containing these serines must be involved in the formation of a specific toxin recognition domain.

Regulation of the transcription of a cluster of Bacillus subtilis spore coat genes.

The pattern of transcription has been examined for a cluster of genes encoding polypeptides some or all of which are assembled into a cross-linked component of the Bacillus subtilis spore coat. Three promoters, designated PVWX, PX and PYZ, were indicated by reverse transcriptase mapping. On the basis of Northern hybridization, it appeared that the cotV, W and X genes were transcribed as a polycistronic mRNA from PVWX as well as a monocistronic cotX mRNA from Px. The cotY and cotZ genes are cotranscribed from the PYZ promoter with a smaller cotY mRNA resulting from premature termination or RNA processing. All four transcripts were synthesized late during sporulation and were not produced in mutants lacking sigma K, which directs RNA polymerase to transcribe genes in the mother-cell compartment of sporulating cells. The DNA-binding protein GerE, which affects transcription of many genes in the mother cell during the late stages of sporulation, was also shown to be involved. There was essentially no cotX mRNA in a gerE mutant and the amounts of cotVWX, cotYZ and cotY mRNAs were somewhat reduced. In vitro run-off transcription studies with sigma K RNA polymerase and GerE confirmed the presence of the three promoters, and directly showed that GerE was necessary for transcription from PX as well as enhanced transcription from the PVWX and PYZ promoters. The DNase I footprints of GerE for all three promoters were immediately upstream of the -35 regions. These GerE binding sites were compared to those in other GerE-responsive promoters and a larger consensus sequence for GerE binding was recognized. This complex transcriptional pattern of the cotVWXYZ cluster is probably necessary to ensure that an optimal amount of each protein is made for the assembly of the spore coat.

Flexibility in the protoxin composition of Bacillus thuringiensis.

In at least three Bacillus thuringiensis subspecies, multiple protoxin genes are confined to just a few of the many plasmids with two or more on one of > 100 mDa and a particular gene, cryIA(b), on a 40-50 mDa plasmid. The latter is unstable but can be maintained in the population by cell mating. Cells which had lost this plasmid compensated by increasing transcription of the remaining protoxin genes resulting in the formation of inclusions which differed from those in the parental strains in their toxicity profiles for selected insects as well as their solubility. Instability of a particular protoxin-encoding plasmid appears to be a mechanism for rapidly shifting the protoxin gene complement and thus the toxicity profiles of these bacteria.

Cloning and characterization of a cluster of genes encoding polypeptides present in the insoluble fraction of the spore coat of Bacillus subtilis.

The Bacillus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 107 degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.

The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival.

Post-exponential Bacillus thuringiensis cells produce both an endospore and a variety of intracellular inclusions. The latter are comprised of protoxins, each being specific for the larvae of certain species from at least three orders of insects. Following ingestion of spores and inclusions, toxicity results in the spores gaining access to haemolymph, a source of nutrients suitable for germination and growth. Most B. thuringiensis subspecies contain multiple, plasmid-encoded protoxin genes, often with several on the same plasmid. These genes have been manipulated in order to understand the basis of toxicity and specificity, information which is important to the use of these toxins as biological control agents. Some protoxin genes are in operons, and others are in close proximity, perhaps to enhance the chances of recombination, and some are on unstable plasmids. The arrangement of these genes is probably important for flexibility in the variety of protoxins packaged into inclusions by a particular subspecies and thus the capacity to adapt to changing populations of insects. Protoxins accumulate over a prolonged period during sporulation because of the sequential transcription from two promoters, each being dependent upon a specific sporulation sigma factor, the relative stability of the messenger RNA, and the synthesis of proteins which stabilize protoxins and perhaps facilitate inclusion assembly. During the post-exponential phase, spore and inclusion formation must be balanced so as to ensure that both are available to contribute to the survival of these bacilli.

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.

Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.

Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein.

The start sites for transcription and translation of a Bacillus subtilis spore coat protein gene, cotT, were determined. The CotT protein was synthesized as a 10.1-kDa precursor which was processed to a coat polypeptide of 7.8 kDa. Insertional inactivation of the cotT gene resulted in spores with an altered appearance of the inner coat layers and slow germination in response to a germination solution containing fructose, glucose, and asparagine. Rates of germination in L-alanine and in Penassay broth were the same as that of the wild type. A strain containing the cotT gene on a low-copy-number plasmid produced spores with an excess of CotT precursor and a thickening of the striated inner coat. These spores responded poorly to all of the germinants mentioned above. A site-directed mutation of the codon at the processing site of CotT resulted in the accumulation of the precursor in sporulating cells and on the spores, but there was no effect on the germination rates or solvent resistance of these spores. Both the lack and the overproduction of CotT led to subtle alterations in the structure of the inner spore coat and in the capacity of spores to respond to particular germinants.

Cloning and analysis of delta-endotoxin genes from Bacillus thuringiensis subsp. alesti.

Bacillus thuringiensis subsp. alesti produced only CryIA(b)-type protoxins, and three cryIA(b) genes were cloned. One was cryptic because of an alteration near the 5' end, and the other two were very similar to each other. The protoxin encoded by one of the latter genes differed from other CryIA(b) protoxins in its greater stability and relative toxicity for two members of the order Lepidoptera.

Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis.

An insect larval toxin designated CryII is produced by several subspecies of Bacillus thuringiensis and differs from the other major delta-endotoxins in these bacteria in its size, toxicity profile and presence as part of an operon with three open reading frames (ORF). Such an operon from a novel B. thuringiensis isolate has been cloned and differs from one previously characterized in the following ways: (a) the size and number of amino acid repeats in one of the ORFs; (b) the smaller size of the CryII protoxin and the presence of a unique 110-kDa CryII-related antigen; and (c) high larvicidal activity for a particular Lepidopteran but low activity for a Dipteran. Various subclones of this operon were introduced into a plasmid-free B. thuringiensis strain and only the cryII gene was found to be necessary for protoxin accumulation.

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.