Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis

Mechold, Undine; Cashel, Michael; Steiner, Kerstin; Gentry, Daniel R.; Malke, Horst

doi:10.1128/jb.178.5.1401-1411.1996

Cited by 116 publications

(138 citation statements)

References 57 publications

Supporting

Mentioning

127

Contrasting

Order By: Relevance

“…In this study, the E. coli RelA/SpoT couple was proposed to act as a toxin/antitoxin system locking cells in a condition of stasis. L. lactis does not possess a SpoT analogRelA exhibits both (p)ppGpp synthesis and degradation activities (16,27). Nevertheless, the stringent response-induced nongrowth state of E. coli and that of Cel − cells of L. lactis appear to have similarities.…”

Section: Discussionmentioning

confidence: 99%

Bet-hedging during bacterial diauxic shift

Solopova

Gestel

Weissing

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

229

245

View full text Add to dashboard Cite

When bacteria grow in a medium with two sugars, they first use the preferred sugar and only then start metabolizing the second one. After the first exponential growth phase, a short lag phase of nongrowth is observed, a period called the diauxie lag phase. It is commonly seen as a phase in which the bacteria prepare themselves to use the second sugar. Here we reveal that, in contrast to the established concept of metabolic adaptation in the lag phase, two stable cell types with alternative metabolic strategies emerge and coexist in a culture of the bacterium Lactococcus lactis. Only one of them continues to grow. The fraction of each metabolic phenotype depends on the level of catabolite repression and the metabolic state-dependent induction of stringent response, as well as on epigenetic cues. Furthermore, we show that the production of alternative metabolic phenotypes potentially entails a bet-hedging strategy. This study sheds new light on phenotypic heterogeneity during various lag phases occurring in microbiology and biotechnology and adjusts the generally accepted explanation of enzymatic adaptation proposed by Monod and shared by scientists for more than half a century.phenotypic heterogeneity | Gram-positive bacteria | metabolic fitness I n nature, bacteria are confronted with a wide range of environmental conditions that change over time. These conditions often elicit specific metabolic responses that increase the division rate of a cell. For example, when bacterial cells are exposed to multiple sugars, they do not metabolize all sugars simultaneously, but rather use the sugar that allows the highest celldivision rate. Cells switch to the less-preferred sugar when the most-preferred one (in many cases glucose) is depleted. Jacques Monod coined this phenomenon "diauxie" (1). Diauxie is characterized by two growth cycles-the first one on the preferred sugar, followed by a second one on the less-preferred sugar. Both are separated by a short period during which the population apparently does not grow. This period is known as the diauxie lag phase. It is typically assumed that cells need time to make the necessary enzymatic adaptations to switch from one substrate to another (2). However, the behavior of individual cells during the lag phase has not been studied in detail. Here, we examine the diauxic shift at the single-cell level in Lactococcus lactis using time-lapse microscopy, in addition to the traditional approach of studying population growth characteristics. Surprisingly, the lag phase at the switch from glucose to cellobiose consumption by L. lactis largely results from the heterogeneous response of cells to the environmental change, rather than the time it takes for cells to make the necessary metabolic adaptations. This result challenges Monod's view of enzymatic adaptation and asks for a revision on the interpretation of population lag phases.

show abstract

Section: Discussionmentioning

confidence: 99%

Bet-hedging during bacterial diauxic shift

Solopova

Gestel

Weissing

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

229

245

View full text Add to dashboard Cite

show abstract

“…Several relA or spoT homologues have been cloned and characterized (Avarbock et al, 1999;Flärdh et al, 1994;Martinez-Costa et al, 1998;Mechold et al, 1996;Ostling et al, 1996;van der Biezen et al, 2000;Wendrich & Marahiel, 1997;Xiao et al, 1991). The induction of relA or spoT homologues is affected by various factors.…”

Section: Discussionmentioning

confidence: 99%

Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

et al. 2005

View full text Add to dashboard Cite

The essential mechanisms and virulence factors enabling Brucella species to survive and replicate inside host macrophages are not fully understood. The authors previously reported that a putative guanosine 59-diphosphate 39-diphosphate (ppGpp) mutant (spoT mutant) of Brucella abortus failed to replicate in HeLa cells. The present study showed that the pattern of surface proteins and morphological change of the spoT mutant were different from B. abortus wild-type. B. abortus wild-type changed its morphology upon treatment with ppGpp synthetase I activation inhibitor. In various tests under stress conditions, including nutrient starvation, nitric oxide resistance, acid resistance and antibiotic resistance, the spoT mutant had a lower stress resistance than B. abortus wild-type. Although the spoT mutant has the same smooth phenotype and LPS profile as B. abortus wild-type, it had a higher rate of adherence to macrophages but lower internalization and intracellular replication within macrophages. The spoT mutant did not co-localize with either late endosomes or lysosomes and was almost cleared from the spleens of mice after 10 days, without splenomegaly. RT-PCR was used to detect spoT mRNA from around 10 6 cells incubated in low-pH enriched medium; it showed that the expression of spoT increased after 30 min incubation. The data suggest that SpoT does not contribute to intracellular trafficking of B. abortus, but contributes to the maintenance of bacterial morphology and the physiological adaptation required for intracellular replication. INTRODUCTIONBrucellosis is a major bacterial zoonosis and an important cause of a serious debilitating disease in humans and abortion and sterility in domestic animals. This disease is caused by species of the genus Brucella, classified in the a2 subdivision of Proteobacteria, small Gram-negative and facultative intracellular pathogens that can multiply within professional and non-professional phagocytes (Delrue et al., 2001;Detileux et al., 1990). The genus Brucella consists of seven species according to antigenic variation and primary host: B. melitensis (sheep and goats), B. suis (hogs), B. abortus (cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (wood rats) and B. maris (marine mammals) (Ko & Splitter, 2003). In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules, thick cell walls, resistance forms or fimbriae and do not show antigenic variation (Finlay & Falkow, 1997).A key aspect of the virulence of Brucella species is their ability to proliferate within professional and nonprofessional phagocytic host cells, therefore successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells (Comerci et al., 2001;Ugalde, 1999). Neither virulence factors that allow intracellular survival of Brucella species nor the specific factors that induce such virulence have been elucidated. B. abortus is inte...

show abstract

“…The E. coli dGTPase (EC 3.1.5.1) catalyzes the Mg 2ϩ -dependent hydrolysis of dGTP to deoxyguanosine and tripolyphosphate (49,50). The major role of the RelA proteins from E. coli and S. equisimilis is the breakdown of (p)ppGpp by a Mn 2ϩ -dependent (p)ppGpp pyrophosphohydrolase activity (51)(52)(53). Very recently, we demonstrated that the HD domain of the E. coli tRNA-nucleotidyltransferase has 2Ј,3Ј-cyclic phosphodiesterase, 2Ј-nucleotidase, and phosphatase activities (54).…”

Section: Screening Of Purified Proteins For Phosphatase Activity-mentioning

confidence: 99%

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Khan

Mizumoto

Ray

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. ). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo-and deoxyribonucleoside 5-monophosphates and ribonucleoside 3-monophosphates with highest affinity to 3-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P 20 -25 ). YfbR was strictly specific to deoxyribonucleoside 5-monophosphates, whereas YjjG showed narrow specificity to 5-dTMP, 5-dUMP, and 5-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di-and triphosphates: YfbR was equally sensitive to both di-and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of biosynthetic and catabolic enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles (1). By opposing the phosphorylation of nucleosides by kinases, intracellular 5Ј-nucleotidases participate in substrate cycles that regulate the cellular levels of ribo-and deoxyribonucleoside monophosphates and thereby all ribo-and deoxyribonucleotide pools (1, 2).Nucleoside monophosphate phosphohydrolases or nucleotidases (EC 3.1.3.5 and EC 3.1.3.6) are phosphatases that specifically dephosphorylate nucleoside monophosphates to nucleosides and inorganic phosphate. Seven mammalian 5Ј-nucleotidases have been identified through cloning and biochemical characterization. These enzymes differ in tissue specificity, subcellular localization, primary structure, and substrate specificity (2). All act on nucleoside monophosphates producing free nucleosides and P i . Each natural nucleotide can be the substrate for several nucleotidases, because they have overlapping specificities. The ubiquitous ecto-5Ј-nucleotidase, eN, 1 is anchored to the surface of the plasma membrane, and AMP is considered to be its major physiological substrate (3). Five other nucleotidases, including dNT-1, occur in the cytosol and one (dNT-2) occurs in mitochondria. dNT-1 and dNT-2 show a preference for the dephosphorylation of dUMP and dTMP (4). The "high K m -nucleotidase" cN-II prefers GMP and IMP (5), the cytosolic nucleotidases cN-IA and cN-IB have a preference for dephosphorylation of AMP (6, 7), and PN-1 (or cN-III) is most active with CMP and UMP (8). For bovine cN-II...

show abstract

Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis

Cited by 116 publications

References 57 publications

Bet-hedging during bacterial diauxic shift

Bet-hedging during bacterial diauxic shift

Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Contact Info

Product

Resources

About