We review the main components of autonomous scientific discovery, and how they lead to the concep... more We review the main components of autonomous scientific discovery, and how they lead to the concept of a Robot Scientist. This is a system which uses techniques from artificial intelligence to automate all aspects of the scientific discovery process: it generates hypotheses from a computer model of the domain, designs experiments to test these hypotheses, runs the physical experiments using robotic systems, analyses and interprets the resulting data, and repeats the cycle. We describe our two prototype Robot Scientists: Adam and Eve. Adam has recently proven the potential of such systems by identifying twelve genes responsible for catalysing specific reactions in the metabolic pathways of the yeast Saccharomyces cerevisiae. This work has been formally recorded in great detail using logic. We argue that the reporting of science needs to become fully formalised and that Robot Scientists can help achieve this. This will make scientific information more reproducible and reusable, and promote the integration of computers in scientific reasoning. We believe the greater automation of both the physical and intellectual aspects of scientific investigations to be essential to the future of science. Greater automation improves the accuracy and reliability of experiments, increases the pace of discovery and, in common with conventional laboratory automation, removes tedious and repetitive tasks from the human scientist.
The basis of science is the hypothetico-deductive method and the recording of experiments in suff... more The basis of science is the hypothetico-deductive method and the recording of experiments in sufficient detail to enable reproducibility. We report the development of Robot Scientist “Adam,” which advances the automation of both. Adam has autonomously generated functional genomics hypotheses about the yeast Saccharomyces cerevisiae and experimentally tested these hypotheses by using laboratory automation. We have confirmed Adam's conclusions through manual experiments. To describe Adam's research, we have developed an ontology and logical language. The resulting formalization involves over 10,000 different research units in a nested treelike structure, 10 levels deep, that relates the 6.6 million biomass measurements to their logical description. This formalization describes how a machine contributed to scientific knowledge.
The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the a... more The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn 1545 mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the fms ( def ) gene, which encodes peptide deformylase (PDF). The C. beijerinckii fms gene product contains four diagnostic residues involved in the Zn 2+ coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from Escherichia coli and Thermus thermophilus , the C. beijerinckii PDF can apparently tolerate N-terminal truncation. The Tn 1545 insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retain...
After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h... more After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ' non-culturable ' cells of the ' Academia ' strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months postinoculation, of a homogeneous population of ostensibly ' non-culturable ' cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10 5 organisms ml N1 , and this value was further increased by one log using supernatant from an actively growing culture. Populations of ' non-culturable ' cells of Mycobacterium tuberculosis were also obtained by the filtration of ' clumpy ' cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ' non-culturable ' cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ' non-culturable ' bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.
Although much is known about the bacterial cellulosome and its various protein components, their ... more Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electrotransformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10 2 transformants per µg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10 5 and 10 7 transformants per µg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.
Journal of molecular microbiology and biotechnology, 2000
The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion seque... more The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific pr...
Journal of molecular microbiology and biotechnology, 2000
Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mut... more Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mutagenesis with Tn1545 and selection for butanol tolerance. It harbours a single copy of Tn 1545 in a 435 bp intergenic region separating two convergently transcribed genes, accC and gldA. The former encodes biotin carboxylase (E.C.6.3.4.14), a subunit of acetyl-CoA carboxylase and the latter encodes glycerol dehydrogenase (E.C.1.1.1.6). Since Tn1545 generates outwardly directed transcripts from its right end, we considered the possibility that the transposon inserted in strain BR54 might affect the expression of the adjacent gldA gene. RT-PCR experiments revealed that the mutant, but not the wild type, contains antisense RNA corresponding to the gldA gene. Correlated with this, the level of glycerol dehydrogenase activity in the mutant was only 25% of that in the wild type when bacteria were grown with either glucose or glycerol as the fermentable substrate. We conclude that transcripts e...
In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by ... more In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation. In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we ha...
Proceedings of the National Academy of Sciences, 1998
Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth ... more Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitation-promoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the viable cell count of dormant M. luteus cultures at least 100-fold and can also stimulate the growth of viable cells. Rpf also stimulates the growth of several other high G+C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii , Mycobacterium smegmatis, and Mycobacterium tuberculosis . Similar genes are widely distributed among high G+C Gram-positive bacteria; genome sequencing has uncovered examples in Mycobacterium leprae and Mb. tuberculosis and others have been detected by hybridization in Mb. smegmatis , Corynebacterium glutamicum, and Streptomyces spp. The mycobacterial gene products may provide different targets for the detection and contro...
Summary Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like protei... more Summary Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like proteins, RpfA‐E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA‐E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a ‘non‐culturable’ state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild‐type organisms confirming that the phenotype was associated with rpf‐like gene loss and that the ‘non‐culturable’ cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent,...
Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five... more Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra‐cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross‐species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bact...
The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpf... more 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-β β β β-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.
Mycobacterium tuberculosis contains five genes, rpfA through rpfE , that bear significant homolog... more Mycobacterium tuberculosis contains five genes, rpfA through rpfE , that bear significant homology to the resuscitation-promoting factor ( rpf ) gene of Micrococcus luteus , whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf -like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf -like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the t...
A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 c... more A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with about 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spoOA plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under the direct control of Spo0A, and additional data are now required to test this prediction.
It has become evident that several of the strains of Clostridium acetobutylicum that have been em... more It has become evident that several of the strains of Clostridium acetobutylicum that have been employed in physiological studies of the acetone-butanol fermentation, are heterogeneous. Studies of the phenotypic and genotypic characteristics of several of these strains (involving inter alia both pyrolysis mass spectrometry and 16s rRNA sequence determinations) demonstrated that the type strain obtained from ATCC was not identical with that supplied by NCIMB, and that NCIMB 8052T is in fact Clostridium beijerinckii. We therefore suggest that the name Clostridium acetobutylicum should be restricted to those strains that are genetically closely related to ATCC 824T (which include strains DSM 792 and DSM 173 1 but not strain P262).
In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positiv... more In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn 1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn 1545 in this strain lies just downstream from gldA , encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydroge...
A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, U... more A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, United Kingdom) with standard isolation techniques. Most of them were putatively assigned to the genera Streptomyces and Micromonospora on the basis of their morphological characteristics, and there appeared to be no difference whether the isolation media contained distilled water or seawater. A group of 20 Micromonospora isolates was selected to undergo further polyphasic taxonomic investigation. Three approaches were used to analyze the diversity of these isolates, 16S rDNA sequencing, fluorescent amplified fragment length polymorphism (AFLP), and Fourier transform infrared spectroscopy (FT-IR). The 16S rDNA sequence analysis confirmed that all of these isolates should be classified to the genus Micromonospora , and they were analyzed with a group of other Micromonospora 16S rDNA sequences available from the Ribosomal Database Project. The relationships of the 20 isolates were observed ...
Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signall... more 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.
The aim of this study was to determine if critical parameters for elite performance could be iden... more The aim of this study was to determine if critical parameters for elite performance could be identified among a population of female shot putters. The performance of seven of the top women shot putters competing at the 2002 USA National Championships was examined. Video data were captured using two Panasonic 60 Hz cameras and the best throws of each athlete were digitized and analyzed using a Peak Motus three-dimensional motion analysis system. Thirty variables were examined for their effect on the distance of the throw. Correlation analysis indicated that measured distance was positively correlated with release speed (r = 0.97, p < 0.0003) and shoulder-hip separation (r = 0.72, p < 0.06) and negatively correlated with release angle (r = -0.74, p < 0.056), rear knee angle at rear foot touchdown (r = -0.93, p < 0.003) and rear knee angle at release (r = -0.76, p < 0.047). Greater knee flexion angle at both rear foot touch down and release along with a neutral shoulder-hip angle at release were identified as the most critical parameters for success among this sample of elite women shot putters. The unique observation about the knee positions at specific events should assist in new training and coaching developments.
We review the main components of autonomous scientific discovery, and how they lead to the concep... more We review the main components of autonomous scientific discovery, and how they lead to the concept of a Robot Scientist. This is a system which uses techniques from artificial intelligence to automate all aspects of the scientific discovery process: it generates hypotheses from a computer model of the domain, designs experiments to test these hypotheses, runs the physical experiments using robotic systems, analyses and interprets the resulting data, and repeats the cycle. We describe our two prototype Robot Scientists: Adam and Eve. Adam has recently proven the potential of such systems by identifying twelve genes responsible for catalysing specific reactions in the metabolic pathways of the yeast Saccharomyces cerevisiae. This work has been formally recorded in great detail using logic. We argue that the reporting of science needs to become fully formalised and that Robot Scientists can help achieve this. This will make scientific information more reproducible and reusable, and promote the integration of computers in scientific reasoning. We believe the greater automation of both the physical and intellectual aspects of scientific investigations to be essential to the future of science. Greater automation improves the accuracy and reliability of experiments, increases the pace of discovery and, in common with conventional laboratory automation, removes tedious and repetitive tasks from the human scientist.
The basis of science is the hypothetico-deductive method and the recording of experiments in suff... more The basis of science is the hypothetico-deductive method and the recording of experiments in sufficient detail to enable reproducibility. We report the development of Robot Scientist “Adam,” which advances the automation of both. Adam has autonomously generated functional genomics hypotheses about the yeast Saccharomyces cerevisiae and experimentally tested these hypotheses by using laboratory automation. We have confirmed Adam's conclusions through manual experiments. To describe Adam's research, we have developed an ontology and logical language. The resulting formalization involves over 10,000 different research units in a nested treelike structure, 10 levels deep, that relates the 6.6 million biomass measurements to their logical description. This formalization describes how a machine contributed to scientific knowledge.
The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the a... more The wild-type strain of Clostridium beijerinckii NCIMB 8052 tends to degenerate (i.e., lose the ability to form solvents) after prolonged periods of laboratory culture. Several Tn 1545 mutants of this organism showing enhanced long-term stability of solvent production were isolated. Four of them harbor identical insertions within the fms ( def ) gene, which encodes peptide deformylase (PDF). The C. beijerinckii fms gene product contains four diagnostic residues involved in the Zn 2+ coordination and catalysis found in all PDFs, but it is unusually small, because it lacks the dispensable disordered C-terminal domain. Unlike previously characterized PDFs from Escherichia coli and Thermus thermophilus , the C. beijerinckii PDF can apparently tolerate N-terminal truncation. The Tn 1545 insertion in the mutants is at a site corresponding to residue 15 of the predicted gene product. This probably removes 23 N-terminal residues from PDF, leaving a 116-residue protein. The mutant PDF retain...
After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h... more After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ' non-culturable ' cells of the ' Academia ' strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months postinoculation, of a homogeneous population of ostensibly ' non-culturable ' cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10 5 organisms ml N1 , and this value was further increased by one log using supernatant from an actively growing culture. Populations of ' non-culturable ' cells of Mycobacterium tuberculosis were also obtained by the filtration of ' clumpy ' cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ' non-culturable ' cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ' non-culturable ' bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.
Although much is known about the bacterial cellulosome and its various protein components, their ... more Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electrotransformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10 2 transformants per µg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10 5 and 10 7 transformants per µg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.
Journal of molecular microbiology and biotechnology, 2000
The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion seque... more The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific pr...
Journal of molecular microbiology and biotechnology, 2000
Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mut... more Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mutagenesis with Tn1545 and selection for butanol tolerance. It harbours a single copy of Tn 1545 in a 435 bp intergenic region separating two convergently transcribed genes, accC and gldA. The former encodes biotin carboxylase (E.C.6.3.4.14), a subunit of acetyl-CoA carboxylase and the latter encodes glycerol dehydrogenase (E.C.1.1.1.6). Since Tn1545 generates outwardly directed transcripts from its right end, we considered the possibility that the transposon inserted in strain BR54 might affect the expression of the adjacent gldA gene. RT-PCR experiments revealed that the mutant, but not the wild type, contains antisense RNA corresponding to the gldA gene. Correlated with this, the level of glycerol dehydrogenase activity in the mutant was only 25% of that in the wild type when bacteria were grown with either glucose or glycerol as the fermentable substrate. We conclude that transcripts e...
In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by ... more In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation. In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we ha...
Proceedings of the National Academy of Sciences, 1998
Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth ... more Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitation-promoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the viable cell count of dormant M. luteus cultures at least 100-fold and can also stimulate the growth of viable cells. Rpf also stimulates the growth of several other high G+C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii , Mycobacterium smegmatis, and Mycobacterium tuberculosis . Similar genes are widely distributed among high G+C Gram-positive bacteria; genome sequencing has uncovered examples in Mycobacterium leprae and Mb. tuberculosis and others have been detected by hybridization in Mb. smegmatis , Corynebacterium glutamicum, and Streptomyces spp. The mycobacterial gene products may provide different targets for the detection and contro...
Summary Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like protei... more Summary Mycobacterium tuberculosis contains five resuscitation‐promoting factor (Rpf)‐like proteins, RpfA‐E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA‐E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a ‘non‐culturable’ state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild‐type organisms confirming that the phenotype was associated with rpf‐like gene loss and that the ‘non‐culturable’ cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent,...
Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five... more Summary Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra‐cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross‐species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bact...
The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpf... more 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-β β β β-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.
Mycobacterium tuberculosis contains five genes, rpfA through rpfE , that bear significant homolog... more Mycobacterium tuberculosis contains five genes, rpfA through rpfE , that bear significant homology to the resuscitation-promoting factor ( rpf ) gene of Micrococcus luteus , whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf -like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf -like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the t...
A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 c... more A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with about 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spoOA plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under the direct control of Spo0A, and additional data are now required to test this prediction.
It has become evident that several of the strains of Clostridium acetobutylicum that have been em... more It has become evident that several of the strains of Clostridium acetobutylicum that have been employed in physiological studies of the acetone-butanol fermentation, are heterogeneous. Studies of the phenotypic and genotypic characteristics of several of these strains (involving inter alia both pyrolysis mass spectrometry and 16s rRNA sequence determinations) demonstrated that the type strain obtained from ATCC was not identical with that supplied by NCIMB, and that NCIMB 8052T is in fact Clostridium beijerinckii. We therefore suggest that the name Clostridium acetobutylicum should be restricted to those strains that are genetically closely related to ATCC 824T (which include strains DSM 792 and DSM 173 1 but not strain P262).
In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positiv... more In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn 1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn 1545 in this strain lies just downstream from gldA , encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydroge...
A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, U... more A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, United Kingdom) with standard isolation techniques. Most of them were putatively assigned to the genera Streptomyces and Micromonospora on the basis of their morphological characteristics, and there appeared to be no difference whether the isolation media contained distilled water or seawater. A group of 20 Micromonospora isolates was selected to undergo further polyphasic taxonomic investigation. Three approaches were used to analyze the diversity of these isolates, 16S rDNA sequencing, fluorescent amplified fragment length polymorphism (AFLP), and Fourier transform infrared spectroscopy (FT-IR). The 16S rDNA sequence analysis confirmed that all of these isolates should be classified to the genus Micromonospora , and they were analyzed with a group of other Micromonospora 16S rDNA sequences available from the Ribosomal Database Project. The relationships of the 20 isolates were observed ...
Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signall... more 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.
The aim of this study was to determine if critical parameters for elite performance could be iden... more The aim of this study was to determine if critical parameters for elite performance could be identified among a population of female shot putters. The performance of seven of the top women shot putters competing at the 2002 USA National Championships was examined. Video data were captured using two Panasonic 60 Hz cameras and the best throws of each athlete were digitized and analyzed using a Peak Motus three-dimensional motion analysis system. Thirty variables were examined for their effect on the distance of the throw. Correlation analysis indicated that measured distance was positively correlated with release speed (r = 0.97, p < 0.0003) and shoulder-hip separation (r = 0.72, p < 0.06) and negatively correlated with release angle (r = -0.74, p < 0.056), rear knee angle at rear foot touchdown (r = -0.93, p < 0.003) and rear knee angle at release (r = -0.76, p < 0.047). Greater knee flexion angle at both rear foot touch down and release along with a neutral shoulder-hip angle at release were identified as the most critical parameters for success among this sample of elite women shot putters. The unique observation about the knee positions at specific events should assist in new training and coaching developments.
Uploads
Papers by Michael Young