Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

Tao, Yi-Ping; Vary, Patricia S.

doi:10.1099/00221287-137-4-797

Cited by 11 publications

(4 citation statements)

References 44 publications

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“…Although this spoIL4C mutant strain initiates sporulation, makes an asymmetric septum, and exhibits forespore nucleoid condensation (normally at t1 to t2 of sporulation), forespore engulfment is blocked (31,32 (30).…”

Section: Resultsmentioning

confidence: 99%

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The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation

et al. 1994

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Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by -0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6) (25). A second parameter which may be involved in spore dormancy is the pH in the spore core, which is rather low, 6.3 for Bacillus cereus spores and 6.4 for Bacillus megaterium spores, when internal pH is measured by distribution of the weak base methylamine (23). In another study, the internal pH of spores of Bacillus subtilis was found to be -6 when measured by 31P nuclear magnetic resonance spectroscopy (1). Although the internal pH of the dormant spore is rather low, it rises about 1 pH unit during spore germination (23,30 (20). The time of entry into sporulation is defined as to, with subsequent hours in sporulation noted as t1, t2, etc. At various times during sporulation, 1-ml samples were taken for assay of glucose dehydrogenase (GDH) and 2-to 4-ml samples were taken for measurement of DPA as previously described (19,20,27). In some cases the condensation of the forespore chromosome was assessed by staining glutaraldehyde-fixed sporulating cells with 4',6-diamidino-2-phenylindole and examining them through an epifluorescence microscope as described previously (22

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Section: Resultsmentioning

confidence: 99%

“…Phone: (203) . Fax: (203) 679-3408. specific accumulation of the large depot of 3-phosphoglyceric acid (3PGA) found in dormant spores (11,25 [31,32]; obtained from P. Vary) were sporulated in SNB medium at 30 or 37°C as previously described (20). The time of entry into sporulation is defined as to, with subsequent hours in sporulation noted as t1, t2, etc.…”

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The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation

et al. 1994

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“…E. coli transformations were carried out using electroporation according to the manufacturer's instructions (Electroporator 2510; Eppendorf). B. megaterium was transformed based on previously published methods, with several modifications (27,30). Twenty-milliliter cultures were grown from 1% inocula of fresh overnight cultures in 250-ml flasks at 35°C and 250 rpm to an optical density (OD) at 660 nm of 0.40.…”

Section: Methodsmentioning

confidence: 99%

PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium

2001

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Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaC Bm ) and PhaR (PhaR Bm ) for activity in vivo and in vitro. PhaC Bm showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR Bm had no obvious homology to PhaE. Previously we showed that PhaC Bm , and here we show that PhaR Bm , is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC Bm to produce an active PHA synthase.

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“…B. megaterium has been extensively studied genetically and is amenable to genetic manipulation (63,66). Hundreds of auxotrophs, division mutants, antibiotic-resistant, and UV-sensitive mutants have been characterized and have been previously mapped in QM B1551 (17,20,31,55,57,62,65). Strain QM B1551 is second only to B. subtilis in the number of multiply marked, characterized strains that are available from the Bacillus Genetic Stock Center (Vary collection, BGSC, Ohio State University; http://www.bgsc.org/).…”

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Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

et al. 2011

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Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B 12 , penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B 12 through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second ␤-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.

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Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

Cited by 11 publications

References 44 publications

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation

PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

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