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Osmotic stress is known to increase the thermotolerance and oxidative-stress resistance of bacteria by a mechanism that is not adequately understood. We probed the cross-regulation of continuous osmotic and heat stress responses by characterizing the effects of external osmolarity (0.3 M versus 0.0 M NaCI) and temperature (43[degrees]C versus 30[degrees]C) on the transcriptome of Escherichia coli K-12. Our most important discovery was that a number of genes in the SoxRS and OxyR oxidative-stress regulons were up-regulated by high osmolarity, high temperature, or a combination of both stresses. This result can explain the previously noted cross-protection of osmotic stress against oxidative and heat stresses. Most of the genes shown in previous studies to be induced during the early phase of adaptation to hyperosmotic shock were found to be also overexpressed under continuous osmotic stress. However, there was a poorer overlap between the heat shock genes that are induced transiently after high temperature shifts and the genes that we found to be chronically up-regulated at 43[degrees]C. Supplementation of the high-osmolarity medium with the osmoprotectant glycine betaine, which reduces the cytoplasmic [K.sup.+] pool, did not lead to a universal reduction in the expression of osmotically induced genes. This finding does not support the hypothesis that [K.sup.+] is the central osmoregulatory signal in Enterobacteriaceae.

Research has been conducted on the bacteria viability in milk after heat treatments. The use of the inducible reporter gene expression system in investigating gene expression in individual cells of viable but non-culturable bacteria after pasteurization is discussed.

Researchers describe a flow cytometry method that clears milk enzymatically of milk proteins and lipids. This provides an accurate count of the bacteria in the milk and the procedure takes less than one hour.

This article comments on the use of the bacterial enzyme savinase for clearing milk proteins before detection and enumeration of Staphylococcus aureus in milk by flow cytometry (Appl. Environ. Microbiol. 66:1228-1232, 2000). Authors' reply to the comment is also presented.

The impact of metal ion bioavailability on the metal content of metallo-beta-lactamase L1 is examined by overexpressing L1 as mature protein (M-L1) and full-length (FL-L1) analogues. Metal analyses on the cytoplasmic and periplasmic fractions of Escherichia coli has shown that the concentration of metal ions in the periplasm is not tightly controlled and has increased as the concentration of metal ions in the growth medium has increased.

In an effort to uncover the role of the high-affinity Zn(II) uptake system in uropathogenic Escherichia coli CFT073, we deleted the znuB gene, which encodes for the transmembrane component of the ZnuABC transporter system. The null mutant for znuB did not grow on minimal medium unless supplemented with excess Zn(II) (50 μM ZnCl2). In contrast, the E. coli K-12 Δ znuB cell line grew well on minimal medium that was not supplemented with Zn(II). The Δ znuB mutant was significantly deficient in the formation of biofilm under static conditions and also showed a substantially reduced migration front of swarm cells. Because motility and biofilm formation are important for E. coli CFT073 pathogenicity, we propose that the high-affinity Zn(II) uptake system may contribute to the virulence of this pathogen in the urinary tract. [ABSTRACT FROM AUTHOR]

In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analogue of L1 was obtained, although this analogue is catalytically inactive. EPR spectra demonstrate the presence of an antiferromagnetically coupled Fe(III)Fe(II) center in Fe-containing L1 and suggest the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly controlled while that of Fe is less so. [ABSTRACT FROM AUTHOR]

Escherichia coli strain BL21 is commonly used as a host strain for protein expression and purification. For structural analysis, proteins are frequently isotopically labeled with deuterium (2H), 13C, or 15N by growing E. coli cultures in a medium containing the appropriate isotope. When large quantities of fully deuterated proteins are required, E. coli is often grown in minimal media with deuterated succinate or acetate as the carbon source because these are less expensive. Despite the widespread use of BL21, we found no data on the effect of different minimal media and carbon sources on BL21 growth. In this study, we assessed the growth behavior of E. coli BL21 in minimal media with different gluconeogenic carbon sources. Though BL21 grew reasonably well on glycerol and pyruvate, it had a prolonged lag-phase on succinate (20 h), acetate (10 h), and fumarate (20 h), attributed to the physiological adaptation of E. coli cells. Wild-type strain NCM3722 (K12) grew well on all the substrates. We also examined the growth of E. coli BL21 in minimal media that differed in their salt composition but not in their source of carbon. The commonly used M9 medium did not support the optimum growth of E. coli BL21 in minimal medium. The addition of ferrous sulphate to M9 medium (otherwise lacking it) increased the growth rate of E. coli cultures and significantly increased their cell density in the stationary phase. [ABSTRACT FROM AUTHOR]

A correction to the article "Growth of E. Coli BL21 in Minimal Media With Different Gluconeogenic Carbon Sources and Salt Contents" is presented.

In an effort to probe whether the metal content of metallo-beta-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analogue of L1 was obtained, although this analogue is catalytically inactive. EPR spectra demonstrate the presence of an antiferromagnetically coupled Fe(III)Fe(II) center in Fe-containing L1 and suggest the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly controlled while that of Fe is less so.