The nucleotide and deduced amino acid sequence of the cationic 19 kDa outer membrane protein OmpH of Yersinia pseudotuberculosis.

The OmpH proteins of enteric bacteria are recently described, small (16 kDa), cationic outer membrane proteins. Because a Yersinia pseudotuberculosis cell envelope protein of this size has been found to cross-react serologically with the human histocompatibility antigen HLA-B27 (B*2701), the sequence of Y. pseudotuberculosis OmpH was determined by sequencing the gene region which encodes mature OmpH. A protein consisting of 143 amino acid residues was found. It was 96% homologous with the OmpH of Y. enterocolitica and 62% homologous with that of Escherichia coli. Two separate OmpH regions had sequence similarity with B*2701; they were identical in both Yersinia species.

Complete sequence of the ompH gene encoding the 16-kDa cationic outer membrane protein of Salmonella typhimurium.

The complete nucleotide sequence of the ompH gene encoding the 16-kDa basic outer membrane protein of Salmonella typhimurium was determined. The OmpH protein is synthesized in a precursor form with additional 20 amino acid residues in the N terminus of the protein. This peptide has common characteristics of signal sequences. The promoter region has strong homology to consensus sequences of Escherichia coli. The expression of ompH was detected in minicells.

The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases.

The Ssc protein, a novel essential protein affecting the function of the enterobacterial outer membrane, matched in a protein homology search best with LpxA (UDP-N-acetylglucosamine 3-hydroxymyristoyl transferase), the enzyme which catalyzes the first step of lipid A biosynthesis. The corresponding genes, located 0.56 kb apart, were 46.7% identical. The search also revealed homology to the bacterial acetyltransferases LacA and NodL, as well as to a hypothetical protein Yglm. The region of residues 109-149 Ssc displayed the highest homology and was also homologous with another bacterial acetyltransferase, CysE, and three other bacterial proteins, two of which are hypothetical. This region and the corresponding regions of all other proteins were found to have a peculiar repeated hexapeptide pattern. Each hexapeptide unit starts with isoleucine (or its equivalent leucine and valine). In most units, the second residue is glycine and the fifth residue either valine or alanine.

Bacterial 'histone-like protein I' (HLP-I) is an outer membrane constituent?

The nucleoid-associated 'histone-like protein I' (HLP-I) protein of E. coli was found to be homologous with the cationic 16-kDa outer membrane protein OmpH of Salmonella typhimurium. Deduced from the nucleotide sequence, the HLP-I protein has 91% identical residues with the OmpH protein. Both proteins have very similar cleavable signal sequences. The nucleotide sequence similarity between the corresponding genes hlpA and ompH is 87%. The ompH gene is located in a gene cluster resembling the hlpA-ORF17 region of E. coli which is close to the Ipx genes involved in the biosynthesis of lipopolysaccharides. The localization of the OmpH/HLP-I protein in the cell is discussed.

Effect of Ssc protein mutations on the outer membrane permeability barrier function in Salmonella typhimurium: a study using ssc mutant alleles made by site-directed mutagenesis.

We have previously discovered and characterized a novel essential enterobacterial protein, the Ssc protein of Salmonella typhimurium and found that the mutation Val291----Met in this protein inhibits bacterial growth at 42 degrees C and the function of its outer membrane permeability barrier at 37 degrees C [7]. In the present paper we prepared, by site-directed mutagenesis, a series of novel plasmid-encoded Ssc mutant proteins and tested their ability to compensate the loss of wild-type Ssc. The mutant proteins Met288----Lys and Gly289----Asp completely lacked this ability, and accordingly, were very defective. Ssc mutants Met288----Leu, Met290----Lys, and Met292----Lys were partially defective. Mutants Met290----Leu and Met292----Leu were non-defective as were also four randomly made mutant proteins with mutations outside the 288-292 region. The S. typhimurium derivative which contained both the chromosomally encoded Ssc Val291----Met and the plasmid-encoded Ssc Gly289----Asp had an outer membrane defect more severe than that caused by SscMet291 only. The mutant Ssc proteins had very little, if any, effect on the outer membrane function in the presence of wild-type Ssc. Even though the function of Ssc is not yet known, our results indicate that region 288-292 is important and that SscAsp289 is thus far the most defective mutant Ssc.

Identification and sequence analysis of the gene mutated in the conditionally lethal outer membrane permeability mutant SS-C of Salmonella typhimurium.

The biosynthesis and structure-function relationships of the enterobacterial outer membrane are subjects of current intensive research. We have previously described the antibiotic supersensitive SS-C mutant (SH7622) of Salmonella typhimurium and shown that its outer membrane permeability barrier against hydrophobic antibiotics is severely defective. In this study, we show that this mutant is heat-sensitive, conditionally lethal, and carries a missense base-pair substitution in a novel gene which we have recently reported and now named the ssc gene. ssc encodes an earlier uncharacterized 36 kd protein (the Ssc protein) and the mutant expresses Ssc which has valine 291 changed to methionine in a methionine-rich region of Ssc. A plasmid containing the wild-type ssc allele completely reverts the antibiotic- and heat-sensitive phenotype of the SS-C mutant. Corresponding plasmids carrying the mutant allele, or an identical mutant allele prepared by localized mutagenesis, are inactive. The ssc gene is probably analogous to the firA locus of Escherichia coli which has earlier been implicated in a totally different function, mRNA synthesis. Furthermore, ssc apparently lies very close to the lpx genes involved in the thus far known steps of lipid A biosynthesis (the distance, approximately 560 bp). To conclude, our findings define a new essential gene involved in the generation of the outer membrane.

The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences.

We have recently described a previously uncharacterized outer membrane protein of Salmonella typhimurium and Escherichia coli and cloned and sequenced the corresponding gene, the ompH gene, of S. typhimurium (P. Koski, M. Rhen, J. Kantele, and M. Vaara, J. Biol. Chem. 264:18973-18980, 1989). We report here the cloning, sequencing, and expression of the corresponding gene of Yersinia enterocolitica. It is significantly homologous to the ompH genes of E. coli and S. typhimurium (homology percentages, 65 and 64%, respectively), has a promoter region strongly homologous to the E. coli 17-bp class consensus promoter, and encodes a protein consisting of 165 amino acids (22 of which form the signal sequence). The plasmid-borne Y. enterocolitica ompH was found to be expressed both in the E. coli host and in minicells. The isolated outer membrane of Y. enterocolitica was shown to contain OmpH. The homology of the Y. enterocolitica OmpH protein is 66% with E. coli OmpH and 64% with S. typhimurium OmpH. All OmpH proteins have almost identical hydrophobic profiles, charge distributions, and predicted secondary structures. Because yersiniae are considered rather distant relatives of E. coli and S. typhimurium in the Enterobacteriaceae family, these results might indicate that most or all strains of the family Enterobacteriaceae have OmpH proteins remarkably homologous to those now sequenced.

Primary structure and expression of the Ssc-protein of Salmonella typhimurium.

A 1020-bp open reading frame (ORF) was found immediately downstream of the ompH gene of Salmonella typhimurium. This ORF (ORF-36) encodes a moderately hydrophobic protein with 341 amino acid residues (calculated molecular mass, 35,928 Da). The ORF-36 product was detected in minicells. Downstream of ORF-36, another ORF was found. It is highly homologous to the E. coli ORF (ORF-17.4) which precedes the lpx-genes involved in lipid A biosynthesis. ORF-36 is probably analogous to the firA gene of E. coli, the sequence of which has not yet been published. Thus it appears that the enterobacterial ompH and lpx genes are separated only by the ORF-36 and ORF-17.4 genes. We also discuss the data on the function of the ORF-36 protein. On this basis, we suggest that the protein could be called the Ssc protein.

Preferential synthesis of heptaacyl lipopolysaccharide by the ssc permeability mutant of Salmonella typhimurium.

In Salmonella typhimurium, a chromosomal gene termed ssc has been shown to cause an antibiotic-supersensitive phenotype. We studied the effect of the ssc gene on the chemical composition of the lipopolysaccharide component, using a thermosensitive ssc1 mutant (SH7622) that grows poorly at 42 degrees C. Analysis of the lipopolysaccharide by various techniques including fast-atom-bombardment mass spectrometry of lipid A, and determination of the type of linkage of fatty acids, revealed a profound temperature-dependent effect associated with the ssc1 mutation. At the non-permissive temperature, SH7622 contained hexadecanoic acid in the majority of lipid A molecules, resulting in the exclusive presence of heptaacyl lipopolysaccharide. This effect was largely reversed by the introduction of the cloned wild-type ssc gene to SH7622 and much reduced by growth of SH7622 at 37 degrees C.

The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines.

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc, converting the acceptor to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Accordingly, we call the recombinant beta1,6-GlcNAc transferase cIGnT6 to emphasize its action at central rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, beta1,4galactosyltransferase and beta1, 3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.