Heterologous expression and kinetic characterisation of Neurospora crassa ß-xylosidase in Pichia pastoris.

To degrade plant hemicelluloses fungi employ ß-xylosidases to hydrolyse xylooligosaccharides, released by endo-xylanases, into xylose. We have expressed the ß-xylosidase from Neurospora crassa in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. The recombinant enzyme is optimally active at 50 °C and pH 5.0 with Km and Vmax values of 8.9 mM and 1052 µmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl ß-xylopyranoside. Xylose is a non-competitive inhibitor with a K(i) of 1.72 mM. The enzyme is characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of ß-1,4 linked xylooligosaccharides (X2, X3 and X4) but also capable of transxylosilation. Catalytic conversion of X2, X3 and X4 decreases (V(max) and k(cat)) with increasing chain length.

Campylobacter bacteriophages and bacteriophage therapy.

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.

Campylobacter succession in broiler chickens.

The competitive ability of Campylobacter coli OR12 over C. jejuni OR1 has been examined in experimental broiler chickens following the observation that C. coli replaced an established C. jejuni intestinal colonisation within commercial chicken flocks reared outdoors [El-Shibiny, A., Connerton, P.L., Connerton, I.F., 2005. Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens. Appl. Environ. Microbiol. 71, 1259-1266]. Co-cultures of C. coli OR12 with C. jejuni OR1, revealed that the two species were able to grow together at similar growth rates in exponential growth phase but if the disparity of the inoculum ratios were >log(10)4 in favour of C. coli OR12, C. jejuni OR1 was observed to prematurely enter decline phase. Chickens were pre-colonised with C. jejuni OR1 at 21-days-old to examine succession in vivo. The birds were inoculated between 2 and 12 days later with C. coli OR12, to determine if the second isolate could efficiently colonise and compete with an established C. jejuni strain. C. coli OR12 were able to co-colonise before replacing C. jejuni OR1 as the dominant species when the birds were more than 27 days of age at the time of administration over a 4-day period. If these criteria were met C. coli OR12 became the dominant isolate otherwise co-colonisation occurred until they were met. C. coli OR12 was also found to displace three alternative C. jejuni strains from pre-colonised chickens challenged with C. coli OR12 at 30 days of age and tested at 40 days. These data raise the possibility of manipulating populations of Campylobacter colonising chickens through competition.

Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens.

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.

Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca.

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log(10) 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log(10) 6.9 CFU/g).

Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens.

Campylobacters and Campylobacter-specific bacteriophages were isolated and enumerated during the rearing cycle of free-range (56 days) and organic chickens (73 days) at 3-day intervals from hatching until slaughter. In both flocks Campylobacter jejuni was the initial colonizer but Campylobacter coli was detected more frequently from 5 weeks of age. The diversity of the Campylobacter isolates was examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA and antimicrobial resistance typing. Bacteriophages were isolated from 51% (19 of 37 birds) of Campylobacter-positive organic birds (log10 2.5 to log10 5.7 PFU/g of cecal contents). The bacteriophages were all typical group III Campylobacter bacteriophages in terms of genomic size but could be characterized in terms of their host range and placed into five different groups. In contrast to the organic birds, anti-Campylobacter activity (bacteriocin-like) was observed in 26% (10 of 38 birds) of Campylobacter-positive free-range birds, and only one bacteriophage was isolated. Appearance of either bacteriophages or anti-Campylobacter activity was associated with changes in the levels of colonization and the predominant genotypes and species isolated. The frequency and potential influence of naturally occurring bacteriophages and/or inhibitory substances on the diversity and fluctuations of populations of campylobacters have not previously been reported in either free-range or organic chickens.

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens.

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.

Identification of a type-D feruloyl esterase from Neurospora crassa.

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.

Functional classification of the microbial feruloyl esterases.

Feruloyl esterases have potential uses over a broad range of applications in the agri-food industries. In recent years, the number of microbial feruloyl esterase activities reported has increased and, in parallel, even more related protein sequences may be discerned in the growing genome databases. Based on substrate utilisation data and supported by primary sequence identity, four sub-classes have been characterised and termed type-A, B, C and D. The proposed sub-classification scheme is discussed in terms of the evolutionary relationships existing between carbohydrate esterases.

A regulator gene for acetate utilisation from Neurospora crassa.

The Neurospora crassa homologue of the Aspergillus nidulans regulatory gene facB has been cloned. The gene encodes a putative transcriptional activator of 865 amino acids that contains a DNA-binding domain with a Zn(II)(2)Cys(6) binuclear cluster, a linker region and a leucine zipper-like heptad repeat. Two internal amino acid sequences are identical to peptide sequences determined from proteolytic fragments of a DNA-binding protein complex specific for genes involved in acetate utilisation and expressed in acetate-induced mycelia of N. crassa. Recombinant expression of the predicted DNA-binding domain demonstrates that it is capable of independent recognition of a subset of the promoter sequences that bind the protein complex from N. crassa. A duplication-induced mutation in the corresponding gene results in an acetate non-utilising phenotype that is characterised by inefficient induction of the enzymes required for acetate utilisation. The new gene does not fall into any existing complementation group and has been designated acu-15.

High-level production of recombinant Aspergillus niger cinnamoyl esterase (FAEA) in the methylotrophic yeast Pichia pastoris.

The cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l(-1) were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.

The Neurospora am gene and NADP-specific glutamate dehydrogenase: mutational sequence changes and functional effects--more mutants and a summary.

A further series of mutant am alleles, encoding potentially active NADP-specific glutamate dehydrogenase (GDH) and capable of complementation in heterocaryons, have been characterized with respect to both GDH properties and DNA sequence changes. Several mutants previously studied, and some of their same-site or second-site revertants, have also been sequenced for the first time. We present a summary of what is known of the properties of all am mutants that have been defined at the sequence level.

Golf complements a GPA1 null mutation in Saccharomyces cerevisiae and functionally couples to the STE2 pheromone receptor.

We have produced a plasmid designed for the expression of heterologous G protein alpha subunits in the yeast Saccharomyces cerevisiae. Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein alpha subunit, to the yeast pheromone response pathway. The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal1- null mutation. A similar response is obtained when an alternative G protein alpha subunit, G(olf), is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni.

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.

Promoter analysis of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa.

Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acu-3) of Neurospora crassa was undertaken. A series of deletions in the 5' non-transcribed region were constructed and the effects of these mutations on the enzyme levels following growth on sucrose and transfer to acetate were measured. Sequences within the region -603 to -271 relative to the transcription start site appear essential for transcription. The region -950 to -1278 is required for sucrose repression, which is consistent with previous protein/DNA gel retardation results of protein extracts from N. crassa cultured on sucrose. Protein extracts from acetate-induced mycelia identify alternative promoter regions apparently involved in acetate-induced gene transcription.

DAB1: a degenerate retrotransposon-like element from Neurospora crassa.

A repeated DNA sequence in the genome of Neurospora crassa has been identified as a family of degenerate retroelements. Retroelements encode protein sequences with clear homology to the reverse transcriptase, RNase H and endonuclease products of the pol genes common to retroviruses and retrotransposons. These sequence comparisons place the N. crassa element within the gypsy group of retrotransposons, akin to other elements found in filamentous fungi. However, the Neurospora element is defective, as no flanking long terminal repeats (LTRs) could be distinguished and the pol gene homologues contain numerous stop codons as a result of multiple base substitutions. The base composition of the element displays significant under-representation of the dinucleotide CpA, the preferred target site of repeat-induced point mutation (RIP). The genomic sequences exhibit G:C to A:T transitions between copies which are diagnostic of RIP. The degenerate retroelement has accordingly been designated by the acronym dab-1 (dead and buried).

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

The Bacillus subtilis 168 chromosome from sspE to katA.

We have cloned and sequenced a 24.5 kb region of the Bacillus subtilis 168 chromosome spanning the sspE and katA genes. The region contains a ribosomal RNA operon, rrnD, a tRNA gene set, trnD and 17 ORFs, 16 with putative ribosome-binding sites. Four of the ORFs (ORF2, ORF14, ORF16 and ORF17) match to known B. subtilis genes (sspE, thiA, senS and katA). Eight of the remaining ORF products show similarities with proteins present in the databases, including an ATP-binding transport protein, a glutamate-1-semialdehyde aminotransferase, a thiol-specific antioxidant protein, a mitomycin radical oxidase and a ferric uptake regulation protein.