Don J. Katcoff

A New Ralstonia Solanacearum High-Affinity Mannose- Binding Lectin Rs-Iil Structurally Resembling the Pseudomonas Aeruginosa Fucose-Specific Lectin Pa- Iil.

The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose. We described previously the properties and sequence of the first lectin, RSL (subunit Mr 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa‐Garber, N., 2002, J Biochem 132: 353–358). The present communication reports the discovery of the second one, RS‐IIL (subunit Mr 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose‐binding lectin PA‐IIL of Pseudomonas aeruginosa. RS‐IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA‐IIL. Determination of the crystal structure of RS‐IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA‐IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918–921). Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand. The binding loop of the cations is fully conserved in RS‐IIL and PA‐IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three‐amino‐acid sequence in the ‘specificity loop’.

Molecular and structural characterization of the HMP-AB gene encoding a pore-forming protein from a clinical isolate of Acinetobacter baumannii.

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.

Genotyping DNA chip for the simultaneous assessment of antibiotic resistance and pathogenic potential of extraintestinal pathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most frequently occurring infections and are mostly caused by extraintestinal pathogenic Escherichia coli. DNA microarrays are potent molecular diagnostic tools for rapid diagnosis of bacterial infections with high relevance for UTIs. In this study, we present the integration and application of two DNA chip modules for the simultaneous detection of single nucleotide polymorphisms in gyrA (quinolone resistance) and fimH (increased adhesion to urinary tract epithelium). The performance of the combined diagnostic chip was assessed by genotyping 140 E. coli strains. Resistance-causing mutations could only be identified in UTI isolates. A complete genotyping assay could be performed in <4h after DNA extraction. Together with the excellent genotyping results, this constitutes a competitive alternative as a standard tool for routine clinical diagnostics.

Yeast linker histone Hho1p is required for efficient RNA polymerase I processivity and transcriptional silencing at the ribosomal DNA

Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by a ChIP experiment showed that Hho1p is required for maximal pol I processivity during rDNA transcription. Psoralen accessibility experiments indicated that Hho1p is necessary for normal rDNA compaction. DNA array expression analysis comparing RNA transcripts in wild-type and hho1 strains before and after a heat-shock showed that Hho1p is necessary to achieve wild-type mRNA levels of transcripts that encode ribosomal components. Taken together, our results suggest that Hho1p is involved in rDNA compaction, and like core histones, is required for efficient rDNA transcription by pol I.

Association of yeast SAP1, a novel member of the ‘AAA’ ATPase family of proteins, with the chromatin protein SIN1

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two‐hybrid system to identify proteins that interact with SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein belonging to the ‘AAA’ family of ATPases, is able to directly interact with SIN1. Furthermore, we show, using recombinant molecules in vitro, that a short 27 amino acid sequence near the N‐terminal of SIN1 is sufficient to bind SAP1. Previous experiments defined different domains of SIN that interact with other proteins and with DNA. The C‐terminal domain of SIN1 was shown to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. The central ‘HMG1‐like region’ of SIN1 binds DNA, while the N‐terminal of SIN1 can bind CDC23, a protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different cellular functions.

Pseudomonas aeruginosa PA-I lectin gene molecular analysis and expression in Escherichia coli.

This communication describes a Pseudomonas aeruginosa DNA fragment (cloned in lambda gt11) which contains the structural gene coding for the galactophilic PA-I lectin (pa-1L, 369 bp) and an additional downstream 237 bp sequence. This DNA is relatively rich in G + C (54%), and exhibits a strong codon preference biased for XXC and also for XXG. The Shine-Dalgarno site of the gene is preceded by an adjacent ATATAT sequence resembling the -10 sequence of the Escherichia coli promoter. The stop codons are followed by a stem and loop structure--typical of the rho-independent transcriptional stop element. This lambda gt11-cloned DNA was expressed in E. coli Y1090 cells. The resulting cell lysates exhibited a galactose-specific hemagglutination and a protein with electrophoretic mobility similar to that of the native PA-I, which were both absent from E. coli lysates infected with ovalbumin gene-bearing bacteriophages. The recombinant PA-I, purified by gel filtration and affinity chromatography, was shown to be a galactophilic hemagglutinin resembling the native lectin in molecular weight and selective reactivity with rabbit anti native PA-I serum. These results are important for development of a safe Pseudomonas aeruginosa vaccine using recombinant DNA techniques, thus avoiding contamination with toxic products of this bacterium.

Cloning and Characterization of the Gene Encoding for OMP-PD Porin: The Major Photobacterium damsela Outer Membrane Protein

The outer membrane protein of Photobacterium damsela (OMP-PD) and the gene encoding for this porin protein were isolated and characterized. The deduced amino acid sequence of the OMP-PD monomer has 338 amino acids and a calculated molecular weight of 36,951 Da. This sequence includes a 22-amino acid signal peptide at the N-terminal, which is not found when the monomer is located in the outer membrane. Native OMP-PD protein forms a trimeric structure of approximately 110 kDa. It exhibits resistance to proteases, and it can be cleaved only following denaturation by SDS. The degree of identity of the OMP-PD amino acid sequence to porins from the Enterobacteriaceae was only 24%. Identity to Vibrio or Photobacterium porins was 38% and 48%, respectively. Nevertheless, the multiple alignment of this sequence with other structurally defined Enterobacteria porins demonstrated that the location of the 16 beta-strands and eight external loops, including a larger external L3 loop, are conserved in OMP-PD. These results, together with the previously known ability of OMP-PD to form an ion channel in artificial liposomes, strongly support its role as a porin in P. damsela and will help further investigations into the role of OMP-PD in P. damsela pathogenicity.

A novel homologue of the prokaryotic htrA gene is differentially expressed in the alga Haematococcus pluvialis following stress

Abstract The alga Haematococcus pluvialis is able to respond to environmental stress by changing from the motile, vegetative green cell form to red stationary aplanospores. This differentiation program is accompanied by dramatic morphological changes that must result, at least in part, from differential gene expression. To begin to identify genes that are differentially expressed as a response to stress, we applied the differential display technique to identify and isolate differentially expressed RNA sequences. Here we report on the isolation and characterization of one such RNA sequence that codes for Haematococcus htrA, a member of a heat shock serine protease family previously described only in prokaryotes. Interestingly, database searches of mouse and human cDNA sequences showed that a previously unreported homologue is also found in a number of tissues. htrA mRNA was not detectable in vegetative cells, but was found at high levels in developing aplanospores. Evidence presented suggests that RNA transcripts encoding this protein are differentially spliced, and that the different splice products are differentially expressed during the developmental process. These experiments provide the groundwork upon which the developmental program of H. pluvialis can be investigated. In addition, they indicate that the htrA family of heat shock serine proteases may play an important role in stress response in higher organisms as well as in bacteria.

The C-terminal domain of SIN1 in yeast interacts with a protein that binds the URS1 region of the yeast HO gene

A protein or protein complex has previously been identified in Saccharomyces cerevisiae which both binds a short DNA sequence in URS1 of HO and interacts with SIN1. SIN1, which has some sequence similarity to mammalian HMG1, is an abundant chromatin protein in yeast and is thought to participate in the transcriptional repression of a specific family of genes. SIN1 binds DNA weakly, though it has no DNA binding specificity. Here we address the nature of the interaction between SIN1 and the specific DNA binding protein(s) to HO DNA. We show that the isolated C-terminal region of SIN1 can interact in vitro with the DNA binding protein, causing a supershift in a gel mobility shift assay. Interestingly, inclusion of the region in SIN1 which contains two acidic sequences, precludes the binding of recombinant protein to the DNA/protein complex.

Characterization of a short unique sequence in the yeast HO gene promoter that regulates HO transcription in a SIN1 dependent manner

Recently it has become clear that general chromatin proteins as well as sequence‐specific DNA binding proteins are important in the control of gene expression. SIN1 in Saccharomyces cerevislae is a chromatin component that regulates the transcription of a family of genes. Previously, we identified a 32 bp unique sequence (here termed XBS) in the promoter of one of those genes, HO, which specifically binds a protein that interacts with SIN1. We also found that this sequence can function as a weak UAS in a heterologous promoter that is dependent on the presence of SIN1. Here we report a relationship between the level of HO expression and the presence of the short sequence in situ in the HO gene. By comparing the expression of HO from wild type or XBS deleted HO promoters, we concluded that XBS serves as a weak UAS in situ in the HO gene, that it influences HO transcription via the SWI/SNF complex, and that sequences other than the XBS mediate the affect of SIN1 on HO transcription. In addition, we show that a portion of the SIN1 protein that has sequence similarity to mammalian HMG1 preferentially binds the XBS.