Antibacterial activity in the sea urchin Echinus esculentus

The studies described in this thesis fall into two parts: 1. A survey of the normal bacterial flora of the common British sea urchin, Echinus esculentus, in comparison with the bacterial flora of seawater and sand from the same locality. 2. An investigation of the antibacterial activity in E. esculentus. This formed the main part of the work. In part 1, the normal bacterial flora, of the sea urchin was examined with isolates from the coelomic fluid, the peristomial membrane and the gut. Aerobic heterotrophic organisms from these sites were identified by a scheme based on that of Shewan, Hobbs and Hodgkiss (1960). The main genera identified were PseudomonasVibrio, Aeromonas, Flavobacterium, Acinetobacter and Moraxella. A few Gram-positive bacteria were also isolated. Of 188 urchins examined, two-thirds had sterile coelomic fluid and it is likely that organisms found there had been introduced by damage to the animal and do not form a permanent indigenous flora. In part 2, initial experiments showed that urchins were capable of clearing, within 24 h, large doses of marine bacteria which had been injected into the coelomic cavity. This indicated that sea urchins possess an efficient antibacterial mechanism. A procedure was developed to examine in vitro the coelomic fluid of sea urchins for antibacterial activity. As test bacterium in these experiments a marine pseudomonad, strain 111, was chosen bacause it produced characteristic black, agar-digesting colonies on marine 2216E agar which were not readily confused with contaminating bacteria. A non-bactericidal control fluid was included in all tests. This consisted of the boiled supernatant of coelomic fluid which was considered to be nutritionally and ionically equivalent to coelomic fluid, and which allowed growth of the test bacterium. Strain 111 incubated in coelomic fluid for 48 h WS'S usually reduced to less than 5% of its initial viable count, whereas in the control fluid the bacteria multiplied. Coelomocytes clot almost immediately when coelomic fluid is withdrawn from urchins but this appeared to have no effect on in vitro antibacterial activity. In vitro the fluid from all 188 urchins studied showed antibacterial activity. The activity was temperature-dependent (optimum

Genetic aspects of antibiotic resistance, haemolysin and bacteriocin production in enterococci

A previous survey of enterococci had identified five strains of Streptococcus faecalis (K55 and SB94) - two subspecies liquefaciens (K60 and K88) and one zymogenes (K87) - and two S. faecium strains (K46 and SB69) which were resistant to tetracycline and streptomycin but susceptible to gentamicin. All the S. faecalis strains and K46 were in addition resistant to erythromycin but only the S. faecium strains were penicillin and ampicillin resistant. The minimal inhibitory concentrations of a further six antibiotics were determined. These values confirmed that in S. faecalis strains, erythromycin resistance was accompanied by resistance to lincomycin and pristinamycin IA, a phenotype typical of macrolide - lincosamide - streptogramin B - type (MLS) antibiotics resistance. The erythromycin resistant K46 however, although resistant to lincomycin, was pristinamycin susceptible and so the basis of resistance is unknown. S. faecalis K60, K87 and SB94 were resistant to kanamycin and neomycin as was S. faecium K46 but all strains were susceptible to spectinomycin. The phenotypes were consistent with resistance mediated by enzymic modification of streptomycin with adenyltransferase (6) and of kanamycin and neomycin with phosphotransferase (3') (5")-III. Erythromycin and tetracycline resistances were expressed constitutively in all strains. Only one S. faecalis (K88) was found to be chloramphenicol resistant and as is typical of Gram-positive bacteria, resistance was inducible. The ability to produce bacteriocin was restricted to beta-haemolytic strain K87 and to strain SB94. Subsequent results indicated that strain K87 probably produced more than one bacteriocin, the activity of which was repressed in the parental strain but which, in derivatives, could be enhanced by the presence of streptomycin. Evidence for the location of resistance, haemolysin and bacteriocin genes was sought from study of the transfer characteristics and stability of markers and from examination of the plasmid content of parental strains and their derivatives. The well characterised S. faecalis subspecies zymogenes strain DS5 (Clewell et al., 1982b) was included for comparison in transfer and curing experiments. All the S. faecalis strains aggregated in response to a cell free filtrate of a plasmid free recipient strain JH2-1, indicating the presence of at least one conjugative plasmid although the low transfer frequencies of most resistance genes in broth matings suggested that response was not necessarily encoded by antibiotic resistance plasmids. Transfer of beta-haemolytic activity and all resistance markers was observed after broth matings but the range of transfer frequencies between strains was wide. Furthermore, the incidence of transfer could be variable particularly in the transfer of DS5 erythromycin resistance and all K87 antibiotic resistances which seemed to be dependent on the production of active donor bacteriocin. Matings of S. faecalis strains carried out on membrane filters were only marginally more efficient in terms of transfer frequencies but were superior with regard to reproducibility of transfer. No antibiotic resistance transfer from S. faecium donors was observed after broth matings and only SB69 tetracycline resistance transferred after filter mating at very low frequency. Several resistance determinants and those encoding β-haemolysin were found to be capable of retransfer indicative of association with genes specifying conjugative ability. Analysis of transconjugant phenotypes revealed that the tetracycline resistance gene of K55, the streptomycin resistance gene of K88 and β-haemolytic activities were always transferred alone but some resistance markers were usually co-transferred with other donor markers. (Abstract shortened by ProQuest.

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate, or oxalate (sulphite could not be tested in uptake assays due to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.Publisher PDFPeer reviewe

Formate-nitrite transporters: Optimisation of expression, purification and analysis of prokaryotic and eukaryotic representatives

The formate-nitrite transporter family is composed of integral membrane proteins that possess six to eight alpha-helical transmembrane domains. Genes encoding these proteins are observed widely in prokaryotic genomes as well as certain groups of lower eukaryotes. Thus far, no structural information is available for this transporter family. Towards this aim, and to provide protein for biophysical studies, overexpression of a prokaryotic (TpNirC, from the hyperthermophilic archaebacterium Thermofilum pendens) and an eukaryotic (AnNitA, from the fungus Aspergillus nidulans) representative was achieved in Escherichia coli and Pichia pastoris hosts, respectively. The proteins were purified to &gt;95% homogeneity yielding quantities sufficient for crystallisation trials and were shown by Circular Dichroism (CD) spectroscopy to have a highly alpha-helical content as expected from in silico predictions. Preliminary investigations by size exclusion chromatography of the oligomeric state of the purified AnNitA protein suggested that it most likely exists as a tetramer. (C) 2009 Elsevier Inc. All rights reserved.</p

Synthetic biology tools for bioprospecting of natural products in eukaryotes

SummaryFilamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast in vivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes

<span style="font-size: 20.5pt;mso-bidi-font-size:13.5pt;font-family:"Times New Roman","serif"">Transformation system of <i><span style="font-size:21.0pt;mso-bidi-font-size:14.0pt; font-family:"Times New Roman","serif"">Beauveria bassiana </span></i><span style="font-size:20.5pt;mso-bidi-font-size:13.5pt;font-family:"Times New Roman","serif"">and <i><span style="font-size:21.0pt;mso-bidi-font-size:14.0pt;font-family: "Times New Roman","serif"">Metarhizium anisopliae </span></i><span style="font-size:20.5pt;mso-bidi-font-size:13.5pt;font-family:"Times New Roman","serif"">using nitrate reductase gene of <i><span style="font-size:21.0pt;mso-bidi-font-size: 14.0pt;font-family:"Times New Roman","serif"">Aspergillus nidulans</span></i> </span></span></span>

650-653<span style="font-size: 15.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">An heterologous transformation system for entomopathogcnic fungi B. bassiana and M. anisopliae was developed based on the use of A. <span style="font-size:15.5pt;mso-bidi-font-size:8.5pt; font-family:" times="" new="" roman","serif""="">nidulans nitrate reductase gene (niaD). <span style="font-size:15.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif""="">B. bassiana <span style="font-size:15.5pt;mso-bidi-font-size:8.5pt;font-family: " times="" new="" roman","serif""="">and <span style="font-size:15.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif""="">M. anisopliae niaD <span style="font-size:15.5pt;mso-bidi-font-size:8.5pt; font-family:" times="" new="" roman","serif""="">stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. <span style="font-size:15.5pt;mso-bidi-font-size:8.5pt; font-family:" times="" new="" roman","serif""="">bassiana and <span style="font-size:16.0pt;mso-bidi-font-size:9.0pt;font-family: " times="" new="" roman","serif""="">M. <span style="font-size:15.5pt; mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif""="">anisopliae at a frequency of 5.8 to 20 transformants per µg of DNA. Most of them were mitotically stable. </span

Comparisons of the <i>Arabidopsis thaliana</i> High-affinity Nitrate Transporter Complex AtNRT2.1/AtNAR2.1 and the <i>Aspergillus nidulans</i> AnNRTA: structure function considerations

<p>The high-affinity nitrate transporter of green plants is composed of two polypeptides, NRT2.1 and NAR2.1, while in fungi it appears that nitrate influx is mediated by NRT2 alone. Another difference between plants and fungi is that the central (cytoplasmic) loop of the 12 membrane spanning regions of NRT is quite large in fungi, consisting of 91 amino acid residues, compared with the relatively short (21 amino acid residues) plant NRT2.1. Here we examine potential amino acid residues involved in the plant NRT2.1:NAR2.1 association by mutation of conserved amino acids in <i>Arabidopsis thaliana</i> AtNRT2.1. Only the replacement of leucine 85 by glutamine disrupted the association between AtNRT2.1 and AtNAR2.1, as examined using the yeast two-hybrid system. Further, to investigate the nitrate-transporting function of AtNRT2.1 in a context free of other members of the NRT2 family, we expressed AtNRT2.1 in <i>Aspergillus nidulans.</i> In the fungal context the plant NRT alone was capable of restoring nitrate transport to a nitrate transport defective mutant, but only when the AtNRT2.1 central loop was replaced by its fungal counterpart.</p

Apparent genetic redundancy facilitates ecological plasticity for nitrate transport

Aspergillus nidulans possesses two high-affinity nitrate transporters, encoded by the nrtA and the nrtB genes. Mutants expressing either gene grew normally on 1–10 mM nitrate as sole nitrogen source, whereas the double mutant failed to grow on nitrate concentrations up to 200 mM. These genes appear to be regulated coordinately in all growth conditions, growth stages and regulatory genetic backgrounds studied. Flux analysis of single gene mutants using (13)NO(3)(–) revealed that K(m) values for the NrtA and NrtB transporters were ∼100 and ∼10 µM, respectively, while V(max) values, though variable according to age, were ∼600 and ∼100 nmol/mg dry weight/h, respectively, in young mycelia. This kinetic differentiation may provide the necessary physiological and ecological plasticity to acquire sufficient nitrate despite highly variable external concentrations. Our results suggest that genes involved in nitrate assimilation may be induced by extracellular sensing of ambient nitrate without obligatory entry into the cell

Expression and antisense inhibition of transgenes in Phytophthora infestans is modulated by choice of promoter and position effects

Procedures were identified for manipulating the expression of genes in the oomycete fungus, Phytophthora infestans. The activities of five putative promoter sequences, derived from the 5' regions of oomycete genes, were measured in transient assays performed in protoplasts and in stable transformants. The sequences tested were from the ham34 and hsp70 genes of Bremia lactucae, the actin-encoding genes of P. infestans and P. megasperma, and a polyubiquitin-encoding gene of P. infestans. Experiments using the GUS reporter gene (encoding beta-glucuronidase) demonstrated that each 5' fragment had promoter activity, but that their activities varied over a greater than tenfold range. Major variation was revealed in the level of transgene expression in individual transformants containing the same promoter::GUS or promoter::lacZ fusion. The level of expression was not simply related to the number of genes present, suggesting that position effects were also influencing expression. Fusions between the ham34 promoter, and full-length and partial GUS genes in the antisense orientation blocked the expression of GUS in protoplasts and in stable transformants