Heterogeneous expression and emulsifying activity of class I hydrophobin from <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Hydrophobins secreted by filamentous fungi self-assemble into an amphipathic film at hydrophilic/hydrophobic interfaces. This unique property suggests that the hydrophobins have a high potential for industrial applications. However, the assemblages of class I hydrophobins are highly insoluble, making such commercial applications difficult. To enhance the solubility of class I hydrophobins, we have attempted to express class I hydrophobin PNH1 from Pholiota nameko fused with glutathione S-transferase (GST) in Escherichia coli. The GST–PNH1 was effectively isolated from the soluble fraction of transformed E. coli, and subsequent analysis revealed that the purified GST–PNH1 had almost the same emulsifying activity as PNH1.     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

The Hydrophobin HCf-1 of Cladosporium fulvum Is Required for Efficient Water-Mediated Dispersal of Conidia

Six hydrophobin genes (HCf-1 to -6) have thus far been identified in the tomato pathogen Cladosporium fulvum. HCf-1 to -4 are Class I hydrophobins and HCf-5 and -6 are Class II hydrophobins. In this paper we describe the isolation of deletion mutants that lack HCf-1, HCf-2, or both these genes. Global down-regulation of the expression of Class I hydrophobins is achieved by homology-dependent gene silencing. Analysis of the mutant strains shows that HCf-1 confers hydrophilic character to the conidia and this facilitates the dissemination of conidia on the surface of water droplets. Other Class I hydrophobins, such as HCf-3 or HCf-4, may be involved in the development and germination of conidia.     

Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7

An in vitro DNA replication system based on extracts prepared from Escherichia coli cells infected with bacteriophage T7 was used to study deletion associated with the repair of double-strand breaks. The gene for T7 ligase was interrupted by a DNA insert which included 17-bp direct repeats. Deletion between the repeats restored the reading frame of the gene, and these DNA molecules could be detected by their ability to give rise to ligase-positive phage after in vitro packaging. T7 genomes that had a pre-existing double-strand break located between the direct repeats were incubated together with intact genomes which had the same direct repeats. Genetic markers placed on either side of the insert in the ligase gene allowed identification of the source of DNA molecules that underwent deletion between the direct repeats. This allowed an assessment of the participation of the molecules with strand breaks in the deletion process, under conditions where any mechanism could contribute to deletion. Approximately three-quarters of the T7 molecules that had lost the region between the direct repeats contained one or both of the partial genomes originally introduced into the reactions. About 50% of the genomes which had undergone deletion had recombined markers between the partial and intact genomes. The data demonstrate that double-strand breaks substantially enhance the contribution of intermolecular recombination to deletion. Received: 19 November 1996 / Accepted: 26 February 1997     

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

Summary The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6–3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%–100% homology with each other.     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.     

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates

Summary The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.     

Genetic control of intrachromosomal recombination

Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

Solution structure and interface‐driven self‐assembly of NC2, a new member of the Class II hydrophobin proteins

Hydrophobins are fungal proteins that self‐assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin‐based applications. Proteins 2014; 82:990–1003. © 2013 Wiley Periodicals, Inc.     

Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3–Cys4 and Cys7–Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSA-coated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7–Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA.     

The Gene Encoding the Heat-Stable Enterotoxin of Vibrio cholerae Is Flanked by 123-Base Pair Direct Repeats

The heat-stable enterotoxin (O1-ST) gene (sto) was cloned from chromosome of the strain GP156 of Vibrio cholerae O1 (Inaba, El Tor) in Escherichia coli K-12, and its nucleotide seqence was determined. The nucleotide sequence of sto was very similar to that of NAG-ST gene (stn) of V. cholerae non-O1. Both sto and stn were flanked by 123-base pair direct repeats which had at least 93% homology to one another and included some inverted repeats. All the strains of V. cholerae, V. mimicus, V. metschnikovii, V. hollisae and Yersinia enterocolitica examined by colony hybridization had the direct repeat sequence regardless of ST-gene possession.     

Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Summary Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 by direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec⁺ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after γ-ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5′ and 3′ ends of dTnp1 together with a perfect palindrome located after the 5′ inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.     

Use of site-specific recombination to regenerate selectable markers

Summary A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 m circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

Hydrophobins from <Emphasis Type="Italic">Aspergillus</Emphasis> species cannot be clearly divided into two classes

Background

Hydrophobins are a family of small secreted proteins with a characteristic pattern of eight cysteine residues found exclusively in filamentous fungi. They have originally been divided into two classes based on their physical properties and hydropathy patterns, and are involved in the attachment of hyphae to hydrophobic structures, the formation of aerial structures and appear to be involved in pathogenicity.

Findings

Analysis of nine genome sequences from seven Aspergilli revealed fifty hydrophobins, where each species displayed between two to eight hydrophobins. Twenty of the identified hydrophobins have not previously been described from these species. Apart from the cysteines, very little amino acid sequence homology was observed. Twenty-three of the identified hydrophobins could be classified as class I hydrophobins based on their conserved cysteine spacing pattern and hydropathy pattern. However twenty-six of the identified hydrophobins were intermediate forms. Notably, a single hydrophobin, ATEG_04730, from Aspergillus terreus displayed class II cysteine spacing and had a class II hydropathy pattern.

Conclusion

Fifty hydrophobins were identified in Aspergillus, all containing the characteristic eight cysteine pattern. Aspergillus terreus exhibited both class I and class II hydrophobins. This is the first report of an Aspergillus species with the potential to express both class I and class II hydrophobins. Many of the identified hydrophobins could not directly be allocated to either class I or class II.

     

High genetic variability of the Streptococcus thermophilus cse central part, a repeat rich region required for full cell segregation activity

The cse gene of Streptococcus thermophilus encodes an extracytoplasmic protein involved in cell segregation. The Cse protein consists of two putative domains: a cell wall attachment LysM domain and a catalytic CHAP domain. These two domains are spaced by an interdomain linker, known as Var-Cse, previously reported to be highly divergent between two S. thermophilus strains. The aim of this study was to assess the extent of this intraspecific variability and the functional involvement of the var-cse region in cell segregation. Analysis of the var-cse sequence of 19 different strains allowed detection of 11 different alleles, varying from 390 bp to 543 bp, all containing interspersed and tandem nucleotides repeats. Overall, 11 different repeat units were identified and some series of these small repeats, named supermotifs, form large repeats. Results suggested that var-cse evolved by deletion of all or part of the repeats and by duplication of repeats or supermotifs. Moreover, sequence analysis of the whole cse locus revealed that the cse ORF is mosaic suggesting that var-cse polymorphism resulted from horizontal transfer. The partial deletion of the var-cse region of the S. thermophilus strain CNRZ368 led to the lengthening of the number of cells per streptococcal chain, indicating that this region is required for full cell segregation in S. thermophilus strain CNRZ368.