- Volume 146, Issue 1, 2000

The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 7·5 to 5·5 that enhances survival at pH 3·0. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses; however, prior adaptation to the other stresses, except heat (42 °C), protected the cells during a subsequent acid challenge at pH 3·5. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 7·5 (37 °C) and then incubated for 30 min under the various stress conditions in the presence of 14C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD% as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 7·5 to 5·5 enhanced synthesis of 64 proteins, 25 of them acid-specific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditons results in complex and diverse alterations in protein synthesis to further cell survival.

It has been demonstrated previously that motile Borrelia burgdorferi cells transform into non-motile cyst-forms when incubated for several weeks in BSKII (a complex medium) lacking rabbit serum. B. burgdorferi cells cannot synthesize fatty acids de novo and serum is thought to provide a source of fatty acids and lipids. When B. burgdorferi cells were serum-starved in defined RPMI medium, ~90% of the cells formed spherical cysts within 48 h. Cyst formation was inhibited by tetracycline. Cyst opening and recovery of vegetative cells was rapidly induced by the addition of either BSKII or rabbit serum. The percentage of viable cells recovered from cysts ranged from 2·9% to 52·5%. Viability was inversely proportional to cyst age. Protein synthesis by B. burgdorferi during serum starvation was examined by labelling cells with Tran35S-Label and analysing the labelled proteins by two-dimensional gel electrophoresis and fluorography. The synthesis of over 20 proteins was induced during serum starvation. Western blots of proteins from vegetative cells and cysts probed with sera from either B. burgdorferi-infected humans or monkeys revealed that several cyst proteins were antigenic. These data suggest that cells of B. burgdorferi, although possessing a small genome and extremely limited biosynthetic capabilities, rapidly respond to conditions of serum starvation by inducing changes in protein synthesis and cell morphology. This study may help explain how cells of B. burgdorferi can survive periods of nutrient deprivation in different hosts and host tissues.

The roles of cyclic formaldehyde oxidation via 6-phosphogluconate dehydrogenase and linear oxidation via the tetrahydromethanopterin (H4MPT)-linked pathway were assessed in an obligate methylotroph, Methylobacillus flagellatus KT, by cloning, sequencing and mutating two chromosomal regions containing genes encoding enzymes specifically involved in these pathways:6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase and methenyl H4MPT cyclohydrolase (gndA, zwf and mch). No null mutants were obtained in gndA or zwf, implying that the cyclic oxidation of formaldehyde is required for C1 metabolism in this obligate methylotroph, probably as the main energy-generating pathway. In contrast, null mutants were generated in mch, indicating that the H4MPT-linked pathway is dispensable. These mutants showed enhanced sensitivity to formaldehyde, suggesting that this pathway plays a secondary physiological role in this methylotroph. This function is in contrast to Methylobacterium extorquens AM1, in which the H4MPT-linked pathway is essential.

Purified glutamate synthase (GOGAT) from Kluyveromyces lactis was characterized as a high-molecular-mass polypeptide, a distinction shared with previously described GOGATs from other eukaryotic micro-organisms. Using degenerate deoxyoligonucleotides, designed from conserved regions of the alfalfa, maize and Escherichia coli GOGAT genes, a 300 bp PCR fragment from the K. lactis GOGAT gene KlGLT1 was obtained. This fragment was used to construct null GOGAT mutants of K. lactis by gene replacement. These mutants showed no growth defect phenotype and were able to grow on ammonium as sole nitrogen source. Double mutants obtained from a cross between a previously described KlGDH1 mutant and the K. lactis null GOGAT strain were full glutamate auxotrophs. These results indicate that glutamate biosynthesis in K. lactis is afforded through the combined action of KlGDH1 and KlGLT1 products.