Arthur I. Aronson

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.

Possible origin and function of the parasporal crystals in Bacillusthuringiensis

Abstract Acrystalliferous strains of Bacillus thuringiensis subsp. kurstaki were isolated at a high frequency following heat treatment of spores. Spores of these strains lacked a 130,000 dalton glycoprotein, the major component common to both parasporal crystals and coats and were nontoxic to tobacco hornworm larvae. Moreover, the deficiency of this glycoprotein resulted in lysozyme sensitivity of the spores of some mutants and the presence of new spore coat proteins in others. All nontoxic acrystalliferous mutants lacked the complete array of at least six plasmids present in the wild type, implying a relationship between presence of plasmid(s) and toxicity. The unique capacity of this species to alter the surface coating of spores which appears to be related to crystal formation may provide flexibility for germination and growth in diverse soil environments.

Inactivation of Bacillus anthracis spores in murine primary macrophages

The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage‐like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome‐associated membrane protein 1 (LAMP1). Expression of a dominant‐negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d‐alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l‐alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

Chromatin-associated proteins of the developing sea urchin embryo. I. Kinetics of synthesis and characterization of non-histone proteins.

Abstract Chromatin-associated non-histone proteins of the sea urchin embryo are heterogeneous, and undergo qualitative as well as quantitative changes throughout early development. The rate of synthesis of these proteins is fairly constant to the pluteus stage and, in contrast to histone synthesis, does not parallel changes in the rates of synthesis of DNA. Evidence for a pool of chromatin-associated nonhistone proteins is provided by following the kinetics of entry into chromatin of labeled protein in pulse-chase experiments of prolonged duration. This pool is synthesized during cleavage and some non-histone proteins continue to associate with chromatin until gastrulation. In addition, different rates of entry of non-histone proteins into chromatin could be detected at different stages.

Biosynthesis of bacterial spore coats

Abstract The biochemical events associated with the assembly of bacterial spore coat proteins have been studied. Pulse-chase experiments using a variety of labeled amino acids have shown that coat precursor protein synthesis begins at the cessation of growth when sporulation commences (early forespore), but several hours prior to the appearance of the morphologically distinct coat structures. 80% of the coat protein can be solubilized with alkaline thioglycollate without disrupting the major morphological components of the coat. In addition, the thioglycollate-insoluble structural components were found to be relatively rich in cystine. The kinetics of cystine incorporation differed from several other amino acids in that there was a four- to fivefold increase of cystine uptake into the coat at the time when the morphological layers appear. Since this incorporation could be selectively inhibited by reagents which compete for sulfhydryl groups, it appeared that cystine was exchanging with available sulfhydryl groups rather than being incorporated into peptide bonds. The polypeptides modified by cystine exchange were preferentially localized in the structural layers and could be solubilized by treatment with sodium lauryl sulfate (resulting in disruption of the coat structure).

Detection of pathogenic E. coli O157:H7 by a hybrid microfluidic SPR and molecular imaging cytometry device.

Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time‐costly growth in cultures. There is need for portable, real‐time, multiplex pathogen detection technology that can predict the safety of food. Surface plasmon resonance (SPR) imaging is a sensitive, label‐free method that can detect the binding of an analyte to a surface by the changes in refractive index that occur upon binding. We have designed a hybrid microfluidic biochip to perform multiplexed detection of single‐celled pathogens using a combination of SPR and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a capture biomolecule targeting a specific pathogen. This biosensor array is enclosed by a polydimethylsiloxane microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. The sample is imaged by SPR on the bottom of the biochip and epi‐fluorescence on the top. The prototype instrument was successfully able to image antibody‐captured E. coli O157:H7 bacteria by SPR and fluorescence imaging. The efficiency of capture of these bacteria by the magnetic particles was determined using spectrophotometric ferric oxide absorbance measurements. The binding of the E. coli to each spot was quantified by measuring the percent of the gold spot area upon which the bacteria was bound and analyzed using NIH ImageJ software. This hybrid imaging approach of pathogenic E. coli detection coupled with an estimate of relative infectivity is shown to be a working example of a testing device for potential foodborne pathogens.

Specificity of Bacillus thuringiensis for lepidopteran larvae: factors involved in vivo and in the structure of a purified protoxin.

The relative LD50 values in two test Lepidoptera of Bacillus thuringiensis subspecies kurstaki HD1, which contains three crylA protoxin genes, was the same as a plasmid‐cured derivative or a Bacillus cereus transcipient containing only one of the three genes. Differential rates of transcription of these genes in the original strain could account, at least partly, for this result. Strains containing only the single protoxin gene (crylA(b)) produced inclusions when grown at 25°C but not 32°C, despite transcription of this gene at both temperatures. The instability of the crylA(b) protoxin was not found in the parental B. thuringiensis subsp. kurstaki HD1 strain grown at either temperature, however, so kurstaki HD1 strains with multiple protoxin genes must produce some stabilizing factor, perhaps another protoxin.

Sequence of the Bacillus subtilis glutamine synthetase gene region

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.

The effect of 5-fluorouracil on bacterial protein and ribonucleic acid synthesis

Abstract 1. 1. In the presence of 5-fluorouracil, a large amount of material accumulates that sediments with the smaller ribosomes. Upon removing the analogue, this material is not converted to larger particles. 2. 2. Fluorouracil has no effect on either the differential rate of incorporation of amino acids nor on the relative amino acid composition of logarithmically growing cells. 3. 3. In magnesium starved bacteria, fluorouracil inhibits both ribosome synthesis and protein synthesis. The effect appears to be directly on ribosome metabolism. 4. 4. The correlation of the selective inhibition of protein synthesis and of ribosome metabolism is discussed.

Membrane-bound messenger RNA and polysomes in sporulating bacteria

By employing DNA hybridization, stable membrane-bound mRNA has been demonstrated in sporulating bacteria. Only membrane-bound polysomes were found in sporulating cells treated with actinomycin. These polysomes have the same size distribution as those in untreated cells and are apparently involved in the synthesis of the structural proteins of spores.