Population heterogeneity in Methylobacterium extorquens AM1

Strovas, Tim; Lidstrom, Mary E.

doi:10.1099/mic.0.025890-0

Cited by 17 publications

(14 citation statements)

References 33 publications

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“…This result is interesting in the light of a series of recent studies reporting phenotypic variation between genetically identical bacteria that live in homogeneous environments (4,9,10,22,24,(26)(27)(28). These differences include cell size, interdivision intervals, gene expression, and behavior.…”

Section: Vol 193 2011 Pole Age Affects M Extorquens Cell Size and mentioning

confidence: 79%

“…extorquens is a facultative methylotroph that is ubiquitous in aquatic and terrestrial environments (15) and is often associated with plants (8). M. extorquens strain AM1 is a genetic model system (31) that shows substantial variation in cell size and interdivision intervals (26,27) between genetically identical cells. Motivated by these reports on phenotypic variation, we asked three questions about cell size asymmetry and the timing of cell division.…”

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Pole Age Affects Cell Size and the Timing of Cell Division in Methylobacterium extorquens AM1

Bergmiller

Ackermann

2011

J Bacteriol

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A number of recent experiments at the single-cell level have shown that genetically identical bacteria that live in homogeneous environments often show a substantial degree of phenotypic variation between cells. Often, this variation is attributed to stochastic aspects of biology-the fact that many biological processes involve small numbers of molecules and are thus inherently variable. However, not all variation between cells needs to be stochastic in nature; one deterministic process that could be important for cell variability in some bacterial species is the age of the cell poles. Working with the alphaproteobacterium Methylobacterium extorquens, we monitored individuals in clonally growing populations over several divisions and determined the pole age, cell size, and interdivision intervals of individual cells. We observed the high levels of variation in cell size and the timing of cell division that have been reported before. A substantial fraction of this variation could be explained by each cell's pole age and the pole age of its mother: cell size increased with increasing pole age, and the interval between cell divisions decreased. A theoretical model predicted that populations governed by such processes will quickly reach a stable distribution of different age and size classes. These results show that the pole age distribution in bacterial populations can contribute substantially to cellular individuality. In addition, they raise questions about functional differences between cells of different ages and the coupling of cell division to cell size.In several species of alphaproteobacteria, the two cells emerging from division have been shown to be systematically different. First, experiments with Caulobacter crescentus and Sinorhizobium meliloti have shown differences in protein localization between cells with new and old poles (13,29). Second, experiments with several species of alphaproteobacteria have shown that cells with "new" poles are smaller than cells with "old" poles (13), (12). Third, in C. crescentus, cells with increasingly old poles show signs of aging, while new pole cells are rejuvenated (2, 3). Here, we used the alphaproteobacterium Methylobacterium extorquens to investigate whether the age of a cell's poles has a systematic effect on its size and on the timing of cell division.M. extorquens is a facultative methylotroph that is ubiquitous in aquatic and terrestrial environments (15) and is often associated with plants (8). M. extorquens strain AM1 is a genetic model system (31) that shows substantial variation in cell size and interdivision intervals (26, 27) between genetically identical cells. Motivated by these reports on phenotypic variation, we asked three questions about cell size asymmetry and the timing of cell division. First, does cell size increase with pole age? Previous experiments with alphaproteobacteria have only distinguished between "old" and "new" poles (12, 13). If we analyze "old" poles of different ages, do we see a systematic effect of pole age on cell size? Secon...

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Section: Vol 193 2011 Pole Age Affects M Extorquens Cell Size and mentioning

confidence: 79%

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confidence: 99%

Pole Age Affects Cell Size and the Timing of Cell Division in Methylobacterium extorquens AM1

Bergmiller

Ackermann

2011

J Bacteriol

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“…Cells were washed once with the corresponding medium without Tet and were resuspended in the same medium at an OD 600 of approximately 0.4 to 0.5. Fluorescence measurements were carried out with a Shimadzu RF-5301PC fluorimeter as described previously (42). Green fluorescent protein (GFP) UV excitation was conducted at 405 nm, and emissions were monitored at 509 nm.…”

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confidence: 99%

“…The 352-bp fragment containing the ectR1-ectA intergenic region was amplified by PCR (the primers are shown in Table S2) and cloned into the pCR2.1 vector. The fragments were subsequently excised by PstI and BamHI and then cloned into the pTSGex promoter probe vector (42), resulting in a construct (pEBP1) containing the respective DNA fragments upstream of the promoterless reporter gene gfp. The resulting constructs were transformed into E. coli S17-1 (39) and then transferred into M. alcaliphilum 20Z and the EBPR01 mutant via conjugation using plasmid pRK2073 as a helper (11).…”

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Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

et al. 2010

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Mineralization of small saline and soda lakes can vary significantly depending on the season and weather conditions. The ability to rapidly adjust intracellular concentrations of key osmolytes (also known as compatible solutes) to changes in external salinity is an important property of microorganisms inhabiting these biotopes (8,18,34). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was found to be a major compatible solute in many halophilic or halotolerant bacteria isolated from alkaline, moderately hypersaline environments (14). This organic solute can be synthesized de novo or taken up from the environment when available (15, 18). The biochemistry and genetics of ectoine synthesis have been described for several bacteria (15,28,29,32). However, little is known about the transcriptional regulation of the ectoine biosynthetic pathway. Comprehensive analysis of the ectoine gene cluster ectABC in Chromohalobacter salexigens showed four putative transcription initiation sites upstream of the ectA start codon. Two 70 -dependent, one S -dependent, and one 32 -dependent promoter were identified and shown to be involved in ectABC transcription in this bacterium (6). Transcription of the ectA, ectB, and ectC genes from Marinococcus halophilus was initiated from three individual 70 / A -dependent promoter sequences located upstream of each gene (3). In Bacillus pasteurii, the ectABC genes are organized in a single operon preceded by a typical A -dependent promoter region (21). The halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z is capable of growth at a salinity as high as 2 M NaCl (19). It was demonstrated that in response to the elevated salinity of the growth medium, M. alcaliphilum cells accumulate ectoine as a major osmoprotective compound (20). The ectoine biosynthesis pathway in M. alcaliphilum 20Z is similar to the pathway employed by halophilic/halotolerant heterotrophs and involves three specific enzymes: diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) (7,21,24,30,32,49). In M. alcaliphilum 20Z, the ectoine biosynthetic genes were shown to be organized in the ectABC-ask operon containing the additional ask gene, encoding aspartokinase (32). Here we describe the transcriptional organization of the ectoine biosynthetic genes in M. alcaliphilum 20Z. We identify a new MarR-like transcriptional regulator (EctR1) and show that EctR1 represses the expression of the ectABC-ask operon from the ectAp 1 promoter by binding at the putative Ϫ10 sequence. These results demonstrate the presence of a new, previously uncharacterized regulatory system for ectoine biosynthesis in the salt-tolerant methanotroph. MATERIALS AND METHODSBacterial strains and culture conditions. The M. alcaliphilum and Escherichia coli strains, plasmids, and primers used in this study are listed in Tables S1 and S2 in the supplemental material. M. alcaliphilum strains were grown at 30°C under a methane-air atmosphere (1:1) or in the presence of 0....

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“…Recently, a surge of evidences found that isogenic populations of microorganisms show substantial cell-to-cell heterogeneity at both cellular and molecular levels (Becskei et al, 2005;Colman-Lerner et al, 2005;Golding et al, 2005;Kelly & Rahn, 1932;Kuang et al, 2004;Le et al, 2005;Lidstrom & Meldrum, 2003;Maloney & Rotman, 1973;Pedraza & van Oudenaarden, 2005;Rosenfeld et al, 2005;Siegele & Hu, 1997;Strovas & Lidstrom, 2009;Strovas et al, 2007). The mechanisms that contribute to this gene expression heterogeneity include intrinsic heterogeneity that exists in biology, noise in the transcription machinery, micro-scale chemical gradients, and genotypic variations because of mutations and selection in individual cells (Stewart & Franklin, 2008).…”

Section: Introductionmentioning

confidence: 97%

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

Shi

Gao

Wang

et al. 2014

Critical Reviews in Biotechnology

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Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.

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Population heterogeneity in Methylobacterium extorquens AM1

Cited by 17 publications

References 33 publications

Pole Age Affects Cell Size and the Timing of Cell Division in Methylobacterium extorquens AM1

Pole Age Affects Cell Size and the Timing of Cell Division in Methylobacterium extorquens AM1

Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices

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