Differentiation between cold shock proteins and cold acclimation proteins in a mesophilic gram-positive bacterium, Enterococcus faecalis JH2-2

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of Ϸ35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (Ϸ22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of Ϸ67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.Shewanella oneidensis MR-1 was first isolated from sediments of Lake Oneida in New York State (20). The cells are gram-negative straight rods capable of moving by means of a single polar flagellum. S. oneidensis MR-1 is a mesophilic, facultative anaerobe having a respiratory type of metabolism, with an optimal growth temperature of Ϸ30°C. Lake Oneida is a shallow freshwater system that freezes over completely during the winter (from early to mid-December to mid-or late January, with complete ice-out in March or April), with ice thickness reaching up to 70 cm (19). During May, water temperatures begin to rise, reaching a maximum of 25°C in midsummer (19).Because of their ability to metabolically reduce metals such as U(VI), Tc(VII), and Cr(VI), organisms such as Shewanella are considered candidates for bioremediation of subsurface metal-contaminated areas. However, subsurface temperatures are often low, and predictions of the utility of Shewanella in such environments require an understanding of the effects of low temperatures on their metabolism and general ecology. Recently, it was shown that S. oneidensis MR-1, while capable of growth at low temperature, exhibited a growth transition at about 10°C, and below these temperatures showed a differential increase in rRNA synthesis compared to DNA synthesis (4). Here we report on other properties of MR-1 grown at low temperature (3°C), including a dramatically different phenotype with changes in morphology, growth rate, ultrastructure, and protein and lipid composition. MATERIALS AND METHODSCells of Shewanella oneidensis strain MR-1 (ATCC 700550) were grown overnight aerobically in batch cultures at 22°C in 150-ml flasks containing 50 ml of Luria-Bertani (LB) broth Miller (Difco) at 130 rpm. For growth measurements, new cultures were started by transferring 1.0 ml of the original culture to a 250-ml Erlenmeyer flask containing 100 ml of LB and incubated at 130 rpm at two temperatures (3 and 22°C). All incubations were done in triplicate. Cells analyzed for proteins and phospholipid fatty acids were harvested by centrifugation and washed with sterile phosphate-buffered saline buffer, wet weight was determined, and the cells were frozen at Ϫ70°C until analysis.The turbidity of the cultures was measured at 600 nm. For measuring cell ...

Section: Discussionsupporting

confidence: 68%

Low-Temperature Growth of Shewanella oneidensis MR-1

Abboud

Popa

Souza-Egipsy

et al. 2005

“…In Streptococcus thermophilus, 24 versus 2 proteins were induced in strains CNRZ302 and PB, respectively (50,36). By 2D electrophoretic analysis, 11 proteins were shown to be induced after a cold stress in E. faecalis (35). Concerning repressed proteins, in L. lactis MG1363, most of them are repressed by salt and about 30 are repressed after a cold shock (53).…”

Section: Discussionmentioning

confidence: 98%

“…After a cold stress, the number of proteins affected differs with the species but also with the methods that are used. In fact, in L. lactis IL1403, 12 proteins are induced even though 22 proteins are induced in L. lactis MG1363 (35,50,51,52). In Streptococcus thermophilus, 24 versus 2 proteins were induced in strains CNRZ302 and PB, respectively (50,36).…”

Section: Discussionmentioning

confidence: 99%

Evidence for Involvement of at Least Six Proteins in Adaptation of Lactobacillus sakei to Cold Temperatures and Addition of NaCl

Marceau

Zagorec

Chaillou

et al. 2004

Lactobacillus sakei is a lactic acid bacterium widely represented in the natural flora of fresh meat. The aim of this study was to analyze the differences in protein expression during environmental changes encountered during technological processes in which L. sakei is involved in order to gain insight into the ability of this species to grow and survive in such environments. Using two-dimensional electrophoresis, we observed significant variation of a set of 21 proteins in cells grown at 4°C or in the presence of 4% NaCl. Six proteins could be identified by determination of their N-terminal sequences, and the corresponding gene clusters were studied. Two proteins belong to carbon metabolic pathways, and four can be clustered as general stress proteins. A phenotype was observed at low temperature for five of the six mutants constructed for these genes. The survival of four mutants during stationary phase at 4°C was affected, and surprisingly, one mutant showed enhanced survival during stationary phase at low temperatures.

“…The overwhelming difference between the growth conditions was the initial pH of the media, however, although it maybe that the difference in growth rate had an effect on the expression of some of the proteins described here and that biofilm or continuous culture grown cells will also express different proteins. Numerous studies have used the high resolving power of 2-D electrophoresis to monitor changes in protein expression during the adaptation of microorganisms to environmental stress (4,22,28,30,41,44). We have used this technique to investigate the regulation of gene product expression of S. oralis grown under conditions of differing pH and used peptide mass fingerprinting to identify those proteins with altered expression.…”

Section: Discussionmentioning

confidence: 99%

Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

Wilkins

Homer

Beighton

2001

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by twodimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.Dental caries is one of the most common causes of tooth loss in the developed world, constituting a substantial economic burden. Following the intake of dietary carbohydrate, acidogenic and aciduric bacteria that form part of the oral biofilm ferment freely metabolizable sugars, producing acid which, when the pH reaches a critical level of below approximately 5.2, results in the demineralization of the tooth. Thus, acidogenicity and aciduricity, the abilities to produce acid and to grow under conditions of low pH, respectively, are considered important virulence determinants for bacteria associated with the initiation and progression of caries. Adaptation to growth at low pH is an essential characteristic for any organism occupying a niche within a caries-prone site or a carious lesion. The acidogenic bacteria which are most closely associated with the initiation and progression of dental caries are mutans streptococci (Streptococcus mutans and Streptococcus sobrinus), lactobacilli, and possibly Actinomyces species, but the role of other bacteria in the progression of the disease has recently been investigated, and a number of studies have highlighted the pathogenic potential of nonmutans streptococci (NMS). Streptococcus oralis, a member of the mitis group of viridans streptococci, which includes Streptococcus pneumoniae, forms a significant proportion of the aciduric microflora of plaque (5), whi...