The metabolic responses of a number of Streptomyces strains to osmotic and heat stress were studied by 13 C nuclear magnetic resonance spectroscopy. During cell growth in a chemically defined medium supplemented with 0.5 M NaCl, tetrahydropyrimidine derivatives (THPs), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and, to a lesser extent, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], were found to accumulate in a significant amount in all bacteria examined. In addition, when the growth temperature was shifted from 30 to 39؇C, the intracellular concentration of THP(A) increased significantly. Moreover, exogenously provided THP(A) or THP(B) or both reversed inhibition of Escherichia coli growth caused by osmotic stress and increased temperature. Although the ability of Streptomyces strains to tolerate high concentrations of NaCl is well known, very little is known about the osmoregulatory strategy in Streptomyces strains. Similarly, the mechanism by which compatible solutes accumulate in a variety of microorganisms is not understood. Our findings suggest the possibility of a novel mechanism of protection of DNA against salt and heat stresses involving the THPs.The ability of Streptomyces strains to tolerate high concentrations of NaCl is well known (24). Living cells adapt to osmotic stress by varying the intracellular concentrations of osmotically active solutes (osmolytes or compatible solutes). These compounds do not produce adverse effects on cellular composition and processes, such as protein structure and solubility, enzyme-substrate interactions, and protein-nucleic acid interactions, as would high concentrations of inorganic salts (reviewed by Low [17]). Intracellular accumulation of osmolytes either by de novo synthesis or by transport from the growth medium confers tolerance to hyperosmotic stress.To date, very little is known about the osmoregulatory strategy of actinomycetes. Because of the transitional nature of the genus Streptomyces (between the simpler eubacteria and the fungi), Harris (8) posed the question of whether actinomycetes use free amino acids (as is characteristic of heterotrophic prokaryotes) or polyols (as is characteristic of heterotrophic eucaryotes) as compatible solutes. Two species of Streptomyces, Streptomyces griseus and S. californicus, were found to intracellularly accumulate mainly proline as a response to hyperosmotic stress (14). Unfortunately, only the intracellular pools of free amino acids were measured in this study. Thus, synthesis of other osmolytes as a response to osmotic stress in Streptomyces strains has not been examined.In a study on the regulation of actinomycin (Act D) biosynthesis by Streptomyces parvulus, two tetrahydropyrimidine derivatives (THPs) were identified (10-12), one a previously unknown metabolite, 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)], and the other 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] (Fig. 1), previously identified (as ectoine) only in halophilic bacteria (6). The...

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5-to 400-fold higher numbers when cultured at a cell mass doubling time (t d ) of 1.8 h than at a slower t d of 11 h. Invasion was optimal when GBS was cultured at a t d of 1.8 h in the presence of >5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t d of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t d of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG)) 2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gelshift assays showed that THP(A) inhibited the EcoRI-(d-(CGCGAATTCGCG)) 2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.Two tetrahydropyrimidine derivatives identified in Streptomyces bacteria (1-3), one a previously unknown metabolite, THP(A), 1 and the other previously identified (as ectoine) in halophilic bacteria (4), THP(B), are now recognized as widely spread osmoprotectants within the bacterial world (5). The role and activities of THPs are of special interest as they represent a limited group of osmoprotectants that are synthesized de novo, in the bacterial cell, in contrast to those transported from the medium (6). Their synthesis in a number of Streptomyces strains as a response to increased salinity and elevated temperature was recently described (7). THPs are small molecules, highly soluble in water and neutral at physiological pH. NMR and x-ray crystallography data show that THPs are zwitterionic molecules with a delocalized charge in the NCN group (Fig. 1) and form the half-chair conformation (8).More information has been accumulated lately on THPs activity in living cells. It was found that exogenously provided ectoine (THP(B)) could reverse growth inhibition, caused by osmotic stress, in Escherichia coli (9), Corynebacterium glutamicum (10), and the soil bacterium Rhizobium meliloti (11). We demonstrated that exogenously provided THP(A), like THP(B), reversed inhibition of E. coli growth by osmotic stress, and moreover, both THP(A) and THP(B) could stimulate growth of E. coli at an elevated temperature (43°C) (7). Recently cloned genes for ectoine synthesis from Halomonas elongata (12) and from Marinococcus halophilus (13) were demonstrated ...

Expression of capsular polysaccharide (CPS) and some surface proteins by group B Streptococcus (GBS) is regulated by growth rate. We hypothesized that precise control of GBS growth, and thus surface-expressed components, could modulate the ability of GBS to invade eukaryotic cells. To test this hypothesis, a dynamic in vitro attachment and invasion system (DIVAS) was developed that combines the advantages of bacterial growth in continuous culture with tissue culture. Tissue culture flasks were modified with inlet and outlet ports to permit perfusion of GBS. Encapsulated type III GBS strains M781 and COH1 and strains COH1-11 and COH1-13 (transposon mutants of COH1 that express an asialo CPS or are acapsular, respectively) were grown in continuous culture in a chemically defined medium at fast mass doubling time (t d ‫؍‬ 1.8 h) and slow (t d ‫؍‬ 11 h) growth rates, conditions previously shown to induce and repress, respectively, type III CPS expression. Encapsulated GBS strains invaded A549 respiratory epithelial cells 20-to 700-fold better at the fast than at the slow growth rate, suggesting a role for CPS. However, unencapsulated GBS were also invasive but only when cultured at the fast growth rate, which indicates that GBS invasion is independent of CPS expression and can be regulated by growth rate. Growth rate-dependent invasion occurred when GBS was grown in continuous culture under glucosedefined, thiamine-defined, and nondefined nutrient limitations. These results suggest a growth rate-dependent regulation of GBS pathogenesis and demonstrate the usefulness of DIVAS as a tool in studies of host-microbe interactions.B acterial attachment to, colonization of, and invasion into host tissue are mediated through a complex series of events that involves changes of surface constituents. Most protocols developed to study attachment and invasion involve growing bacteria in batch culture to a certain phase, placing them onto a monolayer of eukaryotic cells for a specific amount of time, and treating the monolayer to study either attached cells or cells that have invaded, or both.Bacterial growth is a critical, but often overlooked, variable in many types of experiments, including those of attachment and invasion. Although in some reports the effect of the bacterial growth phase on invasiveness in vitro was unequivocally established (1-4), the conclusiveness of the results is curbed by the limitations of the methodology, as growth cannot be controlled by batch culture methods where steady rates of bacterial multiplication occur briefly during the exponential phase and in an ever-changing nutritional environment (5). Moreover, when host-microbe interactions are studied, bacteria placed in a eukaryotic cell culture medium experience a new nutritional environment that may effect expression of certain cell components (6, 7). Indeed, differences in the ability of bacteria to adhere to and invade host tissue may be controlled by surface component expression that is, in turn, regulated by bacterial growth rate and metabolic ...