The amino acid sequence of the egg yolk storage protein phosvitin has been deduced from the nucleotide sequence of part of the chicken vitellogenin gene. Of the phosvitin sequence, 210 amino acids including the N-terminal residue are contained on one large exon, whereas the remaining six amino acids are encoded on the next exon. Phosvitin contains a core region of 99 amino acids, consisting of 80 serines, grouped in runs of maximally 14 residues interspersed by arginines, lysines, and asparagines. The serines of the core region are encoded by AGC and AGT codons exclusively and the arginines by AGA and AGG, which results in a continuous stretch of 99 codons with adenine in the first position. The N-terminal quarter of the phosvitin sequence contains 16 serines grouped in a cluster with alanines and threonines and coded mainly by TCX triplets. The C-terminal part includes 27 serines, preferentially coded by AGC and AGT, 13 histidine residues, and the sequence ...Asn-Gly-Ser... at which the carbohydrate moiety of phosvitin is attached. Heteroduplex formation between cloned DNAs from chicken and Xenopus vitellogenin genes shows that the phosvitin sequence contains a stretch of highly conserved sequence.

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The The outer membranes of gram-negative bacteria obtain their nonspecific permeability for hydrophilic solutes from the presence of porins, a class of proteins that form waterfilled diffusion channels (22). Lack of these proteins leads to resistance to most beta-lactam antibiotics and quinolones (1, 21). In a previous report (28), we described the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that was associated with resistance to beta-lactam antibiotics in transformants both of Escherichia coli and of the parental E. cloacae strain. In these transformants, the amount of the porin proteins OmpF and OmpC present decreased, and an outer membrane protein of approximately 18 kDa was overproduced. In vitro transcription and translation of the recombinant plasmid showed that this protein was encoded by a gene on the cloned fragment. The function of the protein is still unknown, but its overproduction clearly interferes with the presence of OmpF and OmpC in the outer membrane. We propose the name OmpX for this protein, as long as its proper function is obscure. In the accompanying paper (29), the biological characterization of OmpX is presented. In this report, we present the nucleotide sequence of the ompX gene and the deduced amino acid sequence. From these data, some physical properties of OmpX are derived. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains, plasmids, and bacteriophages used are listed in Table 1.Growth conditions. Bacteria were grown at 37°C in brain heart infusion broth (Oxoid Ltd.) supplemented when required with chloramphenicol (34 jig/ml Insertions of the transposable element -yb (11) in plasmid pJS04 were obtained as follows. DNA of plasmid pJS04 was introduced into the F' lac+-containing E. coli CE1304 by transformation. Transformants were selected on Iso-Sensitest agar (Oxoid CM471) containing chloramphenicol (34 ,ug/ml). Several transformants were subsequently mated with E. coli W3110 by filter mating on agar plates for 18 h at 37°C. Cells were collected from the filter, and the mixture was plated on Iso-Sensitest agar plates containing chloramphenicol and nalidixic acid (80 ,ug/ml). Transconjugants were screened subsequently for growth on Iso-Sensitest agar plates containing cephalothin (10 ,g/ml). The positions of the -yb sequence insertions were localized by restriction enzyme digestions.Analysis of plasmid-encoded proteins. Plasmid-encoded proteins were synthesized in vitro in a procaryotic DNAdirected transcription-translation system (Amersham, U.K.

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The outer membrane protein OmpX of Enterobacter cloacae shows high amino acid homology with virulence proteins PagC and Rck from Salmonella typhimurium and with Ail from Yersinia enterocolitica. Here we demonstrate a role for OmpX in the invasion of rabbit ileal tissue by E. cloacae. An organ culture system was used for maintenance of rabbit gut tissue during the experiments. The invasivenesses of three E. cloacae strains, which differed in OmpX content, were compared with each other and with that of Salmonella typhimurium TML (a highly invasive strain) and S. typhimurium LT7 (a noninvasive strain). There was no significant difference between the invasiveness of the wild type and that of an ompX deletion mutant strain of E. cloacae; they were equally as invasive or less invasive than S. typhimurium LT7. The invasiveness of an OmpX overproducer strain of E. cloacae was 10-fold higher than that of its immediate parent carrying only the multicopy plasmid, higher than that of S. typhimurium LT7, but lower than that of S. typhimurium TML. The invasiveness of E. cloacae thus varied directly with the level of OmpX in the outer membrane in rabbit ileal

A chromosomal gentamicin resistance determinant from Pseudomonas aeruginosa was cloned on a 2.4-kb fragment in the broad-host-range vector pLAFR3. Substrate profiles for eight aminoglycosides at three concentrations showed that resistance was due to aminoglycoside- (3)

In order to study the correlation between the presence of aminoglycoside-modifying enzymes in bacteria and the susceptibility of these bacteria to aminoglycosides, 133 resistant strains were collected, representing the most frequently occurring modifying enzymes in clinical isolates today. Enzymes in these resistant strains were identified by the determination of substrate profiles for eight different aminoglycosides in vitro. Thirteen different enzymes or combinations of enzymes appeared to be present in this collection, whereas in seven cases the resistance appeared to be non-enzyme-mediated. The enzyme activities were not reflected in the bacterial susceptibility data for each antibiotic. Cluster analysis of the combined sensitivity data, with biochemical enzyme analysis as a guideline, proved to be unsatisfactory for the identification of aminoglycoside-modifying enzymes in clinical isolates. Much better results were obtained with a stepwise determination scheme. This method relies upon inhibition zone diameters around commercially available sensitivity discs for six aminoglycoside antibiotics. These zone diameters are compared with empirically established critical values, which are characteristic of an enzyme or group of enzymes. By this procedure proper identification of the enzyme(s) proved to be possible in 84% of the 133 resistant strains. For the evaluation of the method 100 consecutively isolated aminoglycoside resistant clinical isolates were analysed by means of the stepwise scheme and the biochemical method. Results were identical for 97 of the 100 strains.

The occurrence of high-level aminoglycoside resistance (HLAmR) was determined for 73 enterococci and 54 group A streptococci by the high-load disc method, tube macrodilution and the polymerase chain reaction (PCR). The PCR method revealed the presence of genes coding for aminoglycoside-3'-O-phosphoryltransferase-III (APH(3')-III), aminoglycoside-6'-N-acetyltransferase/2''-O-phosphoryltransferase (AAC(6')/APH(2'')), or both, in 20.6%, 9.6% and 4.1% of the enterococci, respectively. The prevalence of HLAmR to at least one aminoglycoside among local enterococci was 37% (27/73). Only one of 54 Streptococcus pyogenes isolates produced APH(3')-III and exhibited high-level resistance to kanamycin and streptomycin. In general, the three methods yielded comparable results, with only three discrepancies among the 127 isolates examined. High-load disc screening and tube macrodilution proved to be practical, reliable and reproducible, and thus suitable for routine screening. Of 20 Enterococcus faecalis strains tested, all were penicillin-tolerant. Only one of seven penicillin-tolerant S. pyogenes strains was HLAmR. No association between the two forms of resistance was found.

The nucleotide sequence of the chicken apo Very Low Density Lipoprotein II (apoVLDL II) gene and the regions immediately flanking the gene was determined. Nuclease S1 mapping showed that transcription is initiated at two sites, about 11 bp apart, of which the one lying downstream is used preferentially. Comparison of the 2918-base pair gene sequence with the earlier determined cDNA sequence [Wieringa et al. (1981) Nucleic Acids Research 9, 489-501] enabled us to identify the four exons which are 38 (or 49), 100, 160 and 358 bp long. One of the intron-exon junctions has an unusual sequence. In the 5' flanking region several palindromic sequences are observed. Sequences near the 5' and 3' ends show homologies with the ovalbumin gene.