Interspecific variability of class II hydrophobin GEO1 in the genus Geosmithia

The genus Geosmithia Pitt (Ascomycota: Hypocreales) comprises cosmopolite fungi living in the galleries built by phloeophagous insects. Following the characterization in Geosmithia species 5 of the class II hydrophobin GEO1 and of the corresponding gene, the presence of the geo1 gene was investigated in 26 strains derived from different host plants and geographic locations and representing the whole phylogenetic diversity of the genus. The geo1 gene was detected in all the species tested where it maintained the general organization shown in Geosmithia species 5, comprising three exons and two introns. Size variations were found in both introns and in the first exon, the latter being due to the presence of an intragenic tandem repeat sequence corresponding to a stretch of glycine residues in the deduced proteins. At the amino acid level the deduced proteins had 44.6 % identity and no major differences in the biochemical parameters (pI, GRAVY index, hydropathy plots) were found. GEO1 release in the fungal culture medium was also assessed by turbidimetric assay and SDS-PAGE, and showed high variability between species. The phylogeny based on the geo1 sequences did not correspond to that generated from a neutral marker (ITS rDNA), suggesting that sequence similarities could be influenced by other factors than phylogenetic relatedness, such as the intimacy of the symbiosis with insect vectors. The hypothesis of a strong selection pressure on the geo1 gene was sustained by the low values (<1) of non synonymous to synonymous nucleotide substitutions ratios (Ka/Ks), which suggest that purifying selection might act on this gene. These results are compatible with either a birth-and-death evolution scenario or horizontal transfer of the gene between Geosmithia species.     

HCf-6, a novel class II hydrophobin from Cladosporium fulvum

C. fulvum, a fungal tomato pathogen, has previously been shown to express a complex family of hydrophobin genes including four class I hydrophobins and one class II hydrophobin. Here we describe a gene for HCf-6, a sixth member of the hydrophobin family and the second class II gene. The protein is predicted to consist of a signal sequence, an N-terminus rich in glycine and asparagine and a C-terminal hydrophobic domain which bears the hall-marks of hydrophobins. In contrast to the previously described class II hydrophobin HCf-5, HCf-6 is expressed in mycelium growing in pure culture and mRNA levels do not increase during sporulation. It is down-regulated by carbon starvation but not by depletion of nitrogen in the growth medium.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er.

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

Hydrophobins DGH1, DGH2, and DGH3 in the lichen-forming basidiomycete Dictyonema glabratum

Dictyonema glabratum is a lichen-forming basidiomycete whose symbiotic phenotype shares similarities to both lichens and its non-lichen-forming relatives. In the photobiont layer of D. glabratum intercellular gas-filled spaces are present even when the lichen is water-saturated. The walls of hyphae lining air cavities are covered by a hydrophobic, rodlet-patterned layer, assumed to be formed by hydrophobins. Hot SDS-insoluble, but trifluoroacetic acid-soluble lichen cell wall extracts contained seven proteins. The N-terminal sequence of the most abundant 14-kDa protein was used to carry out cDNA cloning by RT-PCR. The deduced amino acid sequence of the amplified fragment encoded a class I hydrophobin, called DGH1. The cDNA sequence encoding the signal peptide was cloned by RACE-PCR, which also coamplified cDNA fragments encoding two additional class I hydrophobins, DGH2 and DGH3. The three proteins share 54 to 66% amino acid identity. The D. glabratum hydrophobin extract containing either all proteins or primarily DGH1 self-assembled and formed a rodlet mosaic similar to the one observed in situ. Concentration of the protein extract was shown to influence the length of the self-assembled rodlets.     

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.

     

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.     

Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.     

Molecular Cloning and Sequencing of the Extracellular Pectate Lyase II Gene from Erwinia carotovora Er

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for peetate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1 % and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

A Human Rotavirus with Rearranged Genes 7 and 11 Encodes a Modified NSP3 Protein and Suggests an Additional Mechanism for Gene Rearrangement

A human rotavirus (isolate M) with an atypical electropherotype with 14 apparent bands of double-stranded RNA was isolated from a chronically infected immunodeficient child. MA-104 cell culture adaptation showed that the M isolate was a mixture of viruses containing standard genes (M0) or rearranged genes: M1 (containing a rearranged gene 7) and M2 (containing rearranged genes 7 and 11). The rearranged gene 7 of virus M1 (gene 7R) was very unusual because it contained two complete open reading frames (ORF). Moreover, serial propagation of virus M1 in cell culture indicated that gene 7R rapidly evolved, leading to a virus with a deleted gene 7R (gene 7RDelta). Gene 7RDelta coded for a modified NSP3 protein (NSP3m) of 599 amino acids (aa) containing a repetition of aa 8 to 296. The virus M3 (containing gene 7RDelta) was not defective in cell culture and actually produced NSP3m. The rearranged gene 11 (gene 11R) had a more usual pattern, with a partial duplication leading to a normal ORF followed by a long 3' untranslated region. The rearrangement in gene 11R was almost identical to some of those previously described, suggesting that there is a hot spot for gene rearrangements at a specific location on the sequence. It has been suggested that in some cases the existence of short direct repeats could favor the occurrence of rearrangement at a specific site. The computer modeling of gene 7 and 11 mRNAs led us to propose a new mechanism for gene rearrangements in which secondary structures, besides short direct repeats, might facilitate and direct the transfer of the RNA polymerase from the 5' to the 3' end of the plus-strand RNA template during the replication step.     

Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus.

The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage lambda gt11 and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.     

ANT1, an aromatic and neutral amino acid transporter in Arabidopsis

A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine. This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD. Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains. Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members. Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter. RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.     

Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells.

We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11. The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency. A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2. The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa. This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene. The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein. Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin. Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.     

Expression and cellular localization of the protein encoded by the 1C7 gene: a recently described component of the MHC

The major histocompatibility complex (MHC) is located on human Chromosome 6 and includes clusters of class I, class II, and class III genes. Centromeric to the class I region is a cluster of genes designated as MHC class IV encoding genes involved in immunity and inflammation, including the 1C7 gene. The human 1C7 gene has several alternatively spliced forms and potentially codes for proteins with at least three unique carboxy termini. 1C7 mRNA in human (h1C7) is present in spleen, tonsil, B and NK cell lines, and with a different splicing pattern in liver. The 1C7 RNA and protein are present at highest levels in the germinal center of the lymphoid follicles in tonsil. The protein is expressed in NKL cells, tonsil, and unexpectedly in brain. In contrast, the mouse 1C7 gene is transcribed in liver but is predicted to be a pseudogene. However, the 1C7 homologue expressed in rat is predicted to have long stretches of amino acids essentially identical to the human protein.