Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains.

Non-proteolytic, Group II strains of Clostridium botulinum are of particular concern to the food industry because of their ability to survive and grow in REPFEDs (refrigerated processed foods of extended durability). Their analysis would benefit from the availability of a gene transfer system. In the present study we have been able, for the first time, to demonstrate transformation in a representative Group II strain, ATCC 25765. Initial attempts to transform ATCC 25765 with existing clostridial cloning vectors (pMTL540E and pMTL500E) were, however, prevented by a restriction barrier. Through a combination of classical and molecular approaches we were able to show that strain ATCC 25765 possesses a restriction endonuclease (Cbol) and a methylase activity (M. Cbol) which have the same specificity as Mspl and M.Mspl, respectively. Cbol cleaves the palindrome 5'-CCGG-3' to generate a 3'-GC sticky end, whilst M.Cbol specifically methylates the external C residue. An E. coli host was generated which expressed a Bacillus subtilis methylase enzyme (M.BsuF1) with equivalent specificity to M.Cbol. Plasmids (pMTL540E and pMTL500E) prepared in this strain were subsequently shown to be capable of transforming ATCC 25765. The highest frequencies (0.8 X 10(4) transformants per microg of DNA) were obtained when cells were cultivated in media supplemented with 1% (w/v) glycine, and when the electroporation was undertaken at 10 kV/cm, 25 microF and at 400 ohms. Having developed an effective transformation procedure, we went on to construct reporter cassettes based on the Thermanaerobacterium sulfurigenes lacZ and the Vibrio fischeri luxAB genes. Using the former, and promoter regions isolated from the botulinum toxin genes, we have obtained preliminary evidence that reporter genes may be used to evaluate the physiological factors that affect toxin production in the food environment.

Genetic characterisation of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916.

An 8 kb segment of the Clostridium botulinum NCTC 2916 genome 5' to the type A botulinum neurotoxin gene has been sequenced revealing five open reading frames. Four encode components (HA70, HA17, HA34 and NTNH/A) of the progenitor toxin complex. The product of the fifth, OrfX, possesses a putative C-terminal helix-turn-helix motif, exhibits homology with known regulatory proteins (including MsmR from Streptococcus mutans, UviA from C. perfringens and Orftxe1 located upstream of the C. difficile toxin B gene) and is also found within the vicinity of genes encoding tetanus toxin and types B, C, D and G botulinum toxins. Primer extension and Northern blotting analysis demonstrates that the genes are expressed as two divergent operons [HA34, HA17, HA70] and [NTNH/A, type A toxin gene], with the OrfX gene expressed singly. Immediately adjacent to the transcriptional start sites of the HA34 and NTNH/A genes are two highly conserved motifs (5'-ATTTTagGTTTACAAAA-3' and 5'-ATGTTATATgTaA-3'), separated by 12 bp, that span the putative -35 and -10 promoter regions. Homologous sequences occur in the equivalent position relative to the genes at type C botulinum toxin gene and the tetanus toxin gene loci. It is likely that these sequence motifs, together with OrfX, are involved in the co-ordinate expression of the genes encoding the various components of the botulinum toxin complex in groups I, III and IV C. botulinum strains and in that of the tetanus toxin gene.

Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia.

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

Stresses involved in wearing PVC supplied-air suits: a review.

A side effect of the growth and development of industrial technology is the ever increasing number of hostile environments encountered by man. This situation has resulted in the development of impermeable clothing as a barrier protection against the external environment. Unfortunately, although specific government standards concerning the design and engineering requirements of the impermeable suit have been stated, little or no standardized evaluation of the majority of available suits has been attempted. This review was undertaken to provide an in-depth summary of the physiological effects of a PVC protective suit while working. It was concluded that it was necessary to develop a standardized performance test to determine the protective and stressful characteristics of the individual manufacturer's designed suit. Recommendations of specific criteria for the test were obtained.

The physiological consequences of wearing industrial respirators: a review.

The American Industrial Hygiene Association (AIHA) and American Conference of Govermental Industrial Hygienists (ACGIH) Respiratory Protective Devices Manual, published in 1963, has as its primary source of physiological background the work of Silverman and co-workers performed during World War II. The adoption of permanent OSHA standards governing work tasks requiring workers to use respirators has created a need for further evaluation of the physiological effects of wearing a respirator. This review was undertaken to meet the need of an in-depth evaluation of the currently available psychophysiological data. It was concluded that it was of the utmost importance to develop a physiological and psychological medical screening examination to determine the capability of the worker to use a respirator.

Maximal stress test performance while wearing a self-contained breathing apparatus.

Fifteen adult male volunteers (mean age = 31.0 years; eight non-smokers and seven smokers) carried out a Bruce protocol maximal treadmill stress test under four separate conditions. These four conditions were (1) without Scott Air Pak (SAP) respirator; (2) with SAP respirator apparatus but not wearing face mask; (3) with SAP apparatus, wearing face mask in "demand" breathing mode; and (4) with SAP respirator apparatus, wearing face mask in "pressure-demand" breathing mode. The data indicate that the overriding factor in the approximately 20% decrement in work performance during conditions 2, 3, and 4 was related to the weight of the SAP respirator (15.8 kg), P less than 0.05. Only during condition 3 did eight of 15 subjects report lack of air as one reason for termination. Conditions 3 and 4 resulted in a significant decrease in TFore (2.5 degrees C) and was primarily related to the decreased Tinsp which reached a maximum decrement of 9.7 degrees C. Under all conditions the smokers' work time was significantly less than nonsmokers, P less than 0.05.