Variability of the turgor pressure of individual cells of the gram-negative heterotroph Ancylobacter aquaticus.

Cells of Ancylobacter aquaticus were observed under phase microscopy in a chamber to which a measured pressure could be applied. The initial collapse pressure (Ca), i.e., the lowest pressure needed to collapse the most pressure-sensitive gas vesicles, was measured for 69 cells. The cells were taken from cultures in low-density balanced exponential growth, and the experiments were performed quickly so that the bacteria were in a uniform physiological state at the time of measurement. The turgor pressure, Pt, is the difference between the pressure, C, that would cause collapse of vesicles when removed from the cell and Ca. In this paper we focus on the variability of Pt from cell to cell. Part of the observed variability of Ca was due to the variability of the collapse pressure of individual vesicles (standard deviation [SD] = 90 kPa), but because there were about 100 vesicles per cell and because a change in refracted light after the fifth vesicle (approximately) collapsed probably could be detected by the human eye, the pressure would only have an SD of 18.6 kPa due to this type of sampling error. The observed SD of Pt was 42 kPa, indicating that turgor pressure did vary considerably from cell to cell. However, the turgor pressure was independent of cell size. Statistical analysis showed that Pt would decrease 6.9 kPa over a cell cycle, but with too large an SD (19.9 kPa) to be significant. This implies that the observed change in Pt over the cell cycle is not statistically significant.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.     

Characterization of 2,3-dihydroxybenzoic acid from Nocardia asteroides GUH-2.

Culture filtrates of virulent Nocardia asteroides GUH-2 after growth in acetate minimal medium displayed an absorbance maximum at 320 nm. After isolation by polyamide extraction and anion chromatography, a UV-active compound with this absorbance was shown to be 2,3-dihydroxybenzoic acid (DHB) by nuclear magnetic resonance, gas chromatographic, and mass spectrometric techniques. DHB production under several culture conditions was quantified by a standard high-pressure liquid chromatography assay. Under iron deficiency conditions, N. asteroides GUH-2 excreted up to 11 mg of DHB per liter into the culture medium. No DHB was detected when N. asteroides GUH-2 was grown in an iron-rich medium. With the less virulent strain N. asteroides 10905, DHB was not found under any condition tested.     

Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.

The survival of Salmonella montevideo during serum treatment depends on the presence of an O antigen (O-Ag) associated with the lipopolysaccharide molecule. In this organism, the O antigen is a polysaccharide composed of 0 to more than 55 subunits, each containing 4 mannose residues together with glucose and n-acetylglucosamine. We used a mutant strain of S. montevideo that requires exogenous mannose for the synthesis of O-Ag. Lipopolysaccharide (LPS) was prepared from these cells grown under three different conditions where the availability of exogenous mannose was regulated such that the average number of O-Ag units per LPS molecule, the percentage of LPS molecules bearing long O-Ag side chains, and the percentage of lipid A cores bearing O-Ag were all varied. These changes in LPS profiles were monitored on sodium dodecyl sulfate-polyacrylamide gels, and cells with different LPS profiles were tested for their ability to survive treatment with pooled normal human serum. Survival in serum was associated with LPS that contained an average of 4 to 5 O-Ag units per LPS molecule, and 20 to 23% of the LPS molecules had more than 14 O-Ag units per LPS molecule. Serum survival was less clearly associated with the percentage of lipid A cores covered with O-Ag. We propose, based on these data and on previous work, that the O-Ag polysaccharide provides the cell protection from serum killing by sterically hindering access of the C5b-9 complex to the outer membrane and that a critical density of long O-Ag polysaccharide is necessary to provide protection.     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.