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Mitsuaki Moriguchi - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a major fragment of the salt tolerant glutaminase from Micrococcus luteus k 3
Biochemical and Biophysical Research Communications, 2006Co-Authors: Kazuaki Yoshimune, Mamoru Wakayama, Yasuo Shirakihara, Aya Shiratori, Panuwan Chantawannakul, Mitsuaki MoriguchiAbstract:Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48 kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42 kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4 A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.
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Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.
Journal of bioscience and bioengineering, 2005Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuaki MoriguchiAbstract:Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.
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molecular cloning overexpression and purification of Micrococcus luteus k 3 type glutaminase from aspergillus oryzae rib40
Protein Expression and Purification, 2004Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuyo Hoshino, Mitsuaki MoriguchiAbstract:We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.
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isolation and characterization of salt tolerant glutaminases from marine Micrococcus luteus k 3
Journal of Fermentation and Bioengineering, 1994Co-Authors: Mitsuaki Moriguchi, Kenji Sakai, Ryoji Tateyama, Yoichi Furuta, Mamoru WakayamaAbstract:Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases.
Tanetoshi Koyama - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of allylic substrate binding site of Micrococcus luteus b p 26 undecaprenyl diphosphate synthase
Biochemistry, 2003Co-Authors: Keitaro Fujikura, Yuanwei Zhang, Masahiro Fujihashi, Kunio Miki, Tanetoshi KoyamaAbstract:Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al. (2000) J. Biochem. (Tokyo) 128, 917−922] and the existence of a structural P-loop motif for the FPP binding site [Fujihashi, M., et al. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 4337−4342]. To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior. The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintain...
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artificial substrates for undecaprenyl diphosphate synthase from Micrococcus luteus b p 26
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Masahiko Nagaki, Shunsuke Sato, Yuji Maki, Tokuzo Nishino, Tanetoshi KoyamaAbstract:Abstract Substrate specificity of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 was investigated with respect to some alkyl- and bromo-group homologs of isopentenyl diphosphate. Among the homologs relating to the 3-methyl group, but-3-enyl diphosphate ( 2b ) and 3-ethylbut-3-enyl diphosphate ( 3b ) were accepted as substrates, with (all- E )-farnesyl diphosphate (FPP) to give 7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate, and a mixture of 3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and 3,7-diethyleicosa-2,6,10,14,18-pentaenyl diphosphates, respectively. With respect to the homologs modified at the 4 position of isopentenyl diphosphate, (4 E )-3-methylpent-3-enyl diphosphate ( 2f ) was accepted as a substrate to give (4 S )-(2 Z ,6 E ,10 E ,14 E )-4-methylgeranylgeranyl- and (4 S ,8 S )-(2 Z ,6 Z ,10 E ,14 E ,18 E )-4,8-dimethylgeranylfarnesyl diphosphates. Neither (4 Z )-3-methylpent-3-enyl diphosphate nor 4-bromo-3-methylbut-3-enyl diphosphates was accepted as a substrate at all.
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molecular cloning expression and characterization of the genes encoding the two essential protein components of Micrococcus luteus b p 26 hexaprenyl diphosphate synthase
Journal of Bacteriology, 1998Co-Authors: Naoto Shimizu, Tanetoshi Koyama, Kyozo OguraAbstract:ABSTRACT The structural genes encoding the two essential components A and B of hexaprenyl diphosphate synthase, which produce the precursor of the prenyl side chain of menaquinone-6, were cloned from Micrococcus luteus B-P 26.
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purification of solanesyl diphosphate synthase from Micrococcus luteus a new class of prenyltransferase
Journal of Biological Chemistry, 1991Co-Authors: Shinichi Ohnuma, Tanetoshi Koyama, Kyozo OguraAbstract:The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.
Kazuaki Yoshimune - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a major fragment of the salt tolerant glutaminase from Micrococcus luteus k 3
Biochemical and Biophysical Research Communications, 2006Co-Authors: Kazuaki Yoshimune, Mamoru Wakayama, Yasuo Shirakihara, Aya Shiratori, Panuwan Chantawannakul, Mitsuaki MoriguchiAbstract:Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48 kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42 kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4 A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.
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Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.
Journal of bioscience and bioengineering, 2005Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuaki MoriguchiAbstract:Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.
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molecular cloning overexpression and purification of Micrococcus luteus k 3 type glutaminase from aspergillus oryzae rib40
Protein Expression and Purification, 2004Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuyo Hoshino, Mitsuaki MoriguchiAbstract:We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.
Arseny S. Kaprelyants - One of the best experts on this subject based on the ideXlab platform.
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effect of secreted rpf protein on intracellular contacts in Micrococcus luteus and mycobacterium smegmatis cultures
Microbiology, 2011Co-Authors: Vadim D Nikitushkin, Galina R. Demina, Arseny S. KaprelyantsAbstract:The effect of Resuscitation promoting factor (Rpf) on intercellular contacts in the cultures of Micrococcus luteus and Mycobacterium smegmatis was investigated using dynamic light scattering (DLS, photon correlation spectroscopy). During the stationary growth phase, the cells of the tested cultures formed extensive aggregates 100 and 300 μ in size for M. smegmatis and M. luteus, respectively. The number of solitary cells was insignificant. Addition of the recombinant Rpf protein (15 μg/ml) resulted dispersion of cell aggregates and emergence of solitary cells. This effect of Rpf decreased in the presence of nitrophenylthiocyanates (NPTs), specific Rpf inhibitors. Presumably, Rpf is involved in the regulation of intracellular interactions and in biofilm formation.
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structural changes and cellular localization of resuscitation promoting factor in environmental isolates of Micrococcus luteus
Microbial Ecology, 2010Co-Authors: Viktoria Koltunov, Michael Young, Charles L Greenblatt, A V Goncharenko, Galya R Demina, Benjamin Y Klein, Arseny S. KaprelyantsAbstract:Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.
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genome sequence of the fleming strain of Micrococcus luteus a simple free living actinobacterium
Journal of Bacteriology, 2010Co-Authors: Michael Young, Arseny S. Kaprelyants, Harry R Beller, Eebeen Goh, Vladislav Artsatbanov, Govind Chandra, Keith F Chater, Lynn G Dover, Tamar Kahan, Nikos C KyrpidesAbstract:Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content 73%) predicted to encode 2403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 IS elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and fourteen response regulators, indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose, and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.
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Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation
Molecular Microbiology, 2006Co-Authors: Galina V. Mukamolova, Elena G. Salina, Galina R. Demina, Alexander G. Murzin, Arseny S. Kaprelyants, Douglas B Kell, Michael YoungAbstract:The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.
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the rpf gene of Micrococcus luteus encodes an essential secreted growth factor
Molecular Microbiology, 2002Co-Authors: Galina V. Mukamolova, Arseny S. Kaprelyants, Douglas B Kell, Obolbek Turapov, Konstantin Kazarian, M V Telkov, Michael YoungAbstract:Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.
Douglas B Kell - One of the best experts on this subject based on the ideXlab platform.
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Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation
Molecular Microbiology, 2006Co-Authors: Galina V. Mukamolova, Elena G. Salina, Galina R. Demina, Alexander G. Murzin, Arseny S. Kaprelyants, Douglas B Kell, Michael YoungAbstract:The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.
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the rpf gene of Micrococcus luteus encodes an essential secreted growth factor
Molecular Microbiology, 2002Co-Authors: Galina V. Mukamolova, Arseny S. Kaprelyants, Douglas B Kell, Obolbek Turapov, Konstantin Kazarian, M V Telkov, Michael YoungAbstract:Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.
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stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor
Archives of Microbiology, 1999Co-Authors: Galina V. Mukamolova, Douglas B Kell, Svetlana S Kormer, Arseny S. KaprelyantsAbstract:Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria. When washed M. luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium. In the absence of Rpf, there was no increase in colony-forming units for up to 10 days. When the inoculum contained less than 105 cells ml–1, macroscopically observable M. luteus growth was not obtained in succinate minimal medium unless Rpf was added. Incubation of M. luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium. The underestimation of viable cells by the most-probable-number (MPN) method in comparsion with colony-forming units was equivalent to the requirement that at least 105 cells grown on succinate medium, 103 cells from old stationary phase, or approximately 10–500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity. The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units. Thus, a basic principle of microbiology –“one cell-one culture”– may not be applicable in some circumstances in which the metabolic activity of “starter” cells is not sufficient to produce enough autocrine growth factor to support cell multiplication.
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On resuscitation from the dormant state of Micrococcus luteus
Antonie van Leeuwenhoek, 1998Co-Authors: Galina V. Mukamolova, Douglas B Kell, Nataliya D. Yanopolskaya, Arseny S. KaprelyantsAbstract:It has been found previously that a significant number of Micrococcus luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2–7 months can be resuscitated in liquid media which contained (statistically) no initially-viable (colony-forming) cells but which were fortified with sterile supernatant from the late logarithmic phase of batch growth. Here it was found that such resuscitation can be done only within a defined time period after taking the first sample from such cultures, necessarily involving agitation of the cells. The duration of this period depends on the age of the starved culture: cells kept on the bench for 3 months possess a 2 month period of resuscitability while cells starved for 6 months can be resuscitated only within 10 days after the beginning of sampling. It is suggested that the input of oxygen to the starved cultures while they are agitated may exert a negative influence on the cells, since cultures stored in anaerobic conditions (under nitrogen) had a more prolonged ’survival' time. The cells which experienced between 10 and 60 days of starvation on the bench could be resuscitated, although the number of resuscitable cells depended strongly on the concentration of yeast extract in the resuscitation medium. This concentration for cells stored on the bench for more than 2 months was 0.05% while ’1-month-old‘ cells displayed a maximum resuscitability in the presence of 0.01% of yeast extract. Application of the fluorescent probe propidium iodide revealed the formation of cells with a damaged permeability barrier if resuscitation was performed by using concentrations of yeast extract of 0.1% and above. Thus the successful resuscitation of bacterial cultures under laboratory conditions may need rather strictly defined parameters if it is to be successfully performed for the majority of cells in a population.
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biochemical changes accompanying the long term starvation of Micrococcus luteus cells in spent growth medium
Archives of Microbiology, 1995Co-Authors: Galina V. Mukamolova, Arseny S. Kaprelyants, N D Yanopolskaya, Tatyana V Votyakova, V I Popov, Douglas B KellAbstract:Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.