The state of autotrophic ethanol production in Cyanobacteria.

Ethanol production directly from CO2 , utilizing genetically engineered photosynthetic cyanobacteria as a biocatalyst, offers significant potential as a renewable and sustainable source of biofuel. Despite the current absence of a commercially successful production system, significant resources have been deployed to realize this goal. Utilizing the pyruvate decarboxylase from Zymomonas species, metabolically derived pyruvate can be converted to ethanol. This review of both peer-reviewed and patent literature focuses on the genetic modifications utilized for metabolic engineering and the resultant effect on ethanol yield. Gene dosage, induced expression and cassette optimizat-ion have been analyzed to optimize production, with production rates of 0·1-0·5 g L(-1) day(-1) being achieved. The current 'toolbox' of molecular manipulations and future directions focusing on applicability, addressing the primary challenges facing commercialization of cyanobacterial technologies are discussed.

Differentiating the growing nosocomial infectious threats Ralstonia pickettii and Ralstonia insidiosa.

Differentiation of the growing nosocomial infectious threats, Ralstonia pickettii and Ralstonia insidiosa, based on nitrate reduction, desferrioxamine susceptibility, arabinose, N-acetyl-glucosamine and phenylacetate assimilation is described. These tests can be used for preliminary identification of Ralstonia pickettii and Ralstonia insidiosa resulting in more accurate identification of these species.

Introducing principles of validation into biochemistry and biotechnology courses: Practical concepts and a framework for active learning.

Although undergraduate biochemistry and biotechnology courses teach the concept of accuracy and precision during practical laboratory sessions, the formal teaching of validation methodologies receives little attention. An increasing number of biochemistry and biotechnology graduates are finding work in industry in the area of industrial validation associated with biopharmaceutical, diagnostics, biomedical device, and pharmaceutical validation. We have introduced a structured introduction to validation into our undergraduate industrial biochemistry programme to illustrate the importance of validation within a framework of good manufacturing practice (GMP) and to show how validation is essential for regulatory compliance.

Ralstonia pickettii in environmental biotechnology: potential and applications.

Xenobiotic pollutants such as toluene and trichloroethylene are released into the environment by various industrial processes. Ralstonia pickettii possess significant biotechnological potential in the field of bioremediation and has demonstrated the ability to breakdown many of these toxic substances. Here, we provide a description of the major compounds that various strains of R. pickettii are capable of degrading and a brief review of their breakdown pathways and an argument for its use in bioremediation.

Ralstonia pickettii: a persistent gram-negative nosocomial infectious organism.

Non-fermenting Gram-negative bacilli create a significant problem in clinical settings, being a widespread cause of nosocomial infections. They are opportunistic pathogens that take advantage of underlying conditions and diseases. Ralstonia pickettii, a non-fermenting Gram-negative bacillus, is regarded as being of minor clinical significance; however, many instances of infections with this organism are reported in the literature. Infections can include bacteraemia/septicaemia caused by contaminated solutions, e.g. distilled water, water for injection and aqueous chlorhexidine solutions. Cases of pseudobacteraemia have been recorded in association with R. pickettii, as have many cases of unusual infections, some of which were very invasive and severe, e.g. meningitis, septic arthritis and osteomyelitis. Six cases of death in four separate instances have also been recorded related to R. pickettii. This review illustrates that R. pickettii is a more important pathogen than was thought previously.

Pulsed field gel electrophoresis to rapidly detect the presence of IncJ conjugative transposon-like elements.

AIMS: To develop a screening method to detect the presence of the IncJ group of integrating conjugative transposon-like elements upon transfer to Escherichia coli. METHODS AND RESULTS: The unique insertion site of known IncJ elements, the prfC gene, is located in a region of the E. coli chromosome between 98.5 and 100 min on the E. coli genetic map. Using pulsed field gel electrophoresis and the rare cutting restriction enzymes SfiI and XbaI insertions of IncJ elements and an estimate of their size could be determined physically. CONCLUSIONS: This method allows initial screening of putative IncJ conjugative transposon-like elements by physical determination of their integration. SIGNIFICANCE AND IMPACT OF THE STUDY: IncJ-like elements, which appear to be highly homologous to the prototype IncJ element R391, have been found associated with recent epidemic outbreaks of cholera in a number of locations worldwide. Because of their integrative biology this method provides the first initial screening method to physically determine their presence upon transfer to E. coli.

The effect of choice of sterilisation method on the biocompatibility and biodegradability of SIS (small intestinal submucosa).

SIS (small intestinal submucosa) is a 3D extracellular matrix (ECM) material of porcine origin. It has a complex composition predominantly composed of collagen type I. SIS is rapidly absorbed, supports early and abundant new vessel growth, and serves as a template for the reconstructive remodelling of several body tissues. Currently SIS products are sterilised using ethylene oxide, gamma irradiation and e-beam irradiation. It is not known how they affect the materials properties such as structure, mechanical strength and biocompatibility. This study investigated the influence of each sterilisation method on the biocompatibility and biodegradation of SIS using L929 mouse fibroblasts. SIS samples were sterilised by each of the above methods under standard conditions. The samples were subjected to hydrolytic degradation conditions for specific periods of time. All sterilisation methods resulted in an increase in the rate of sample degradation. The study indicated that over time e-beam irradiation caused the greatest % weight loss. Applying sample extracts to L929 mouse fibroblasts assessed the biocompatibility of the degradation products. The % cellular protein and % metabolic activity were then assessed using the BCA assay and MTT assay, respectively. All SIS samples caused an increase in both cellular protein production and metabolic activity. Initially samples sterilised by ETO had the greatest effect but this decreased after 28 days. Unsterile samples were found to have a slower more prolonged influence. It is thought that the components released may include extractable growth factors and further studies are required to confirm this.

The role of conjugative transposons in the Enterobacteriaceae.

Although widely studied in gram-positive Streptococci and in the gram-negative Bacteroides, there is a scarcity of information on the occurrence and nature of conjugative transposon-like elements in the well-studied Enterobacteriaceae. In fact, some of the major reviews on conjugative transposons prior to 1996 failed to mention their occurrence in this group. Recently, their presence has been reported in Salmonella, Vibrio and Proteus species, and in some cases such as the SXT element in Vibrio and the IncJ group element CTnR391, there has been some molecular characterization. The elements thus far examined appear to be larger than the common gram-positive conjugative transposons and to be mosaic in structure, with genes derived from several sources. Recent evidence suggests that in the Enterobacteriaceae the elements may be related to enteric pathogenicity islands. The evolution, distribution and role of these elements in the Enterobacteriaceae is discussed.

Isolation and characterisation of the ylmE homologue of Thermus thermophilus.

Screening of a Thermus thermophilus genomic library led to the identification of a homologue of the ylmE gene. ylmE is highly conserved in widely divergent organisms from prokaryotes to mammals, suggesting an important, albeit currently unknown, cellular function. The 633 bp gene has a GC content of 69.2% overall and 90% in the third nucleotide position, while the gene product is predicted to be a soluble cytoplasmic protein of 23,441 Da. It belongs to a family of conserved proteins of unknown function and exhibits amino acid identities ranging from 45% to 28% to the Aquifex aeolicus and Saccharomyces cerevisiae family members, respectively. We speculate that the gene product may be involved in a cellular stress response in T. thermophilus.

Cloning and characterisation of the Hint homologue of the thermophile Thermus thermophilus.

Screening of a genomic library of the thermophile Thermus thermophilus revealed a novel thermophilic hint gene, homologues of which are highly conserved in genera from archaea to mammals. Hint belongs to the HIT protein super-family, which contains two broad groups, Fhit, associated with tumour suppression in eukaryotes and Hint with putatitively protein kinase C inhibitory activity. In T. thermophilus the 321 bp gene has a GC content of 67% overall and 94.4% in the third nucleotide position, with unusually no thymine as a wobble base. The gene product, a small highly conserved 11,996 Da predicted soluble cytoplasmic protein, offers an ideal opportunity to investigate thermostabilising amino acid substitutions. Here we report on the characterisation of the novel hint sequence.

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility.

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.

Monitoring of chromosomal insertions of the IncJ elements R391 and R997 in Escherichia coli K-12.

The integration site(s) of the IncJ element, R391, was localised to a specific region of the Escherichia coli chromosome, between the uxuA and serB loci (98.0-99.5 min), using classical Hfr mapping techniques. F-prime plasmid hosts, diploid for regions spanning the E. coli chromosome, were used as recipients in R391 and R997 conjugal transfer assays. Analysis of transconjugants revealed the integration of R391 and R997 into specific F-primes that contain the uxuA to serB region, but not F-primes that contain other regions of the chromosome. A comparison of the electrophoretic mobility of the original F-primes with those containing inserts demonstrated the integration of large elements, in excess of 85 kb. Linear integration of the IncJ elements into chromosomal DNA was demonstrated in recombination-deficient (recA) backgrounds in the absence of detectable autonomous stages. These observations account for the inability to isolate plasmid DNA from IncJ hosts, and suggests that the elements exhibit a conjugative transposon-like biology in E. coli.

Influence of sodium oxide content on bioactive glass properties.

The rate of in vivo degradation and level of bioactivity of bioactive glasses are composition dependent [1]. By altering bioactive glass composition, the rate of resorption can be controlled. The network connectivity of a glass can be used to predict various physical properties of the glass including its solubility and, hence, its bioactivity [2]. Glass solubility increases as network connectivity is reduced. Glasses in the soda-lime phosphosilicate system were studied. The initial choice of composition was based on phosphate content and low network connectivity. A systematic substitution of calcium oxide for sodium oxide on a molar basis was made in order to examine the influence of sodium oxide content on the glass properties while keeping the network connectivity constant. The glass transition temperature and the peak crystallization temperature were seen to decrease linearly with increasing sodium oxide content. Thermal expansion coefficient and glass density were also seen to be related to sodium oxide content. Preliminary in vitro biocompatibility studies revealed that the glasses of higher sodium oxide content were associated with a cytotoxic response. The measurement of media pH indicated that this cytotoxic effect was due to ion exchange reactions at the glass surface.

Transfer of the IncJ plasmid R391 to recombination deficient Escherichia coli K12: evidence that R391 behaves as a conjugal transposon.

A study of the IncJ plasmid R391 confirmed a low frequency of transfer between recombination proficient (recA+) Escherichia coli (10(-5) donor -1). Reanalysis of its transfer to recombination deficient (recA) E. coli revealed an equivalent transfer frequency to and from all mutants tested. Extrachromosomal DNA could not be detected in either recA+ or recA transconjugants, while R391 proved refractory to curing in both backgrounds implying a high degree of stability. The integration of R391 into a specific region of the chromosome was demonstrated by its transfer as part of the exogenote mobilised from the transfer origins of Hfr strains BW6165 and JC158. Transfer of R391 coupled to recA independent chromosomal integration has significant implications as to the nature and classification of the element. We propose that R391 behaves like a conjugal transposon.

Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391.

The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA. Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv-resistant derivatives. This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222. A recombinant plasmid containing both functions (KanR and Uvs+) was obtained. The uv-sensitizing function was mapped to a 4-kb EcoRI fragment.

The effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation.

The presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA+ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

Cloning in Bacillus subtilis of an extremely thermostable alpha amylase: comparison with other cloned heatstable alpha amylases.

A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RPO1 into Bacillus subtilis. Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B. licheniformis FDO2 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans. The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases.

cis-Platinum(II)diamminodichloride-induced mutagenesis in E. coli K12: crowding depression of mutagenesis.

Cis-Platinum(II)diamminodichloride (cis-PDD)-induced mutations to prototrophy were studied in Escherichia coli. Mutagenesis was not detected in a recA nor in a lexA mutant, but was greater in a uvrA strain than in a repair-proficient strain, at a given treatment of cis-PDD. Increasing the plating density above 10(5) cells per plate did not give an equivalent increase in revertants per plate [crowding depression of mutagenesis (Bockrath et al., 1980)]. Growth rates were similar at different plating densities and crowding depression of mutagenesis was observed in both excision-proficient and excision-deficient strains. A filtrate of a plate wash from crowded plates, of either treated or untreated cultures, further reduced the mutation frequencies over that due to crowding depression of mutagenesis.