| 1. |
- Ederth, Josefine, et al.
(författare)
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A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 37:2, s. e15-
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Tidskriftsartikel (refereegranskat)abstract
- With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.
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| 2. |
- Ghosh, Jaydip, et al.
(författare)
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Sporulation in mycobacteria
- 2009
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 106:26, s. 10781-10786
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Tidskriftsartikel (refereegranskat)
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| 3. |
- Ghosh, Jaydip, et al.
(författare)
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Sporulation in mycobacteria
- 2009
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 106:26, s. 10781-10786
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette-Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.
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| 4. |
- Kirsebom, Leif A, et al.
(författare)
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New Vaccines
- 2009
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Patent (populärvet., debatt m.m.)
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| 5. |
- Lesterlin, Christian, et al.
(författare)
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Asymmetry of Chromosome Replichores Renders the DNA Translocase Activity of FtsK Essential for Cell Division and Cell Shape Maintenance in Escherichia coli
- 2008
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Ingår i: PLOS GENET. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 4:12, s. e1000288-
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.
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| 6. |
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| 7. |
- Pillay, Radhamani, et al.
(författare)
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An Improved Redundancy Scheme for the Optimal Utilization of Onboard Computers
- 2009
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Ingår i: Proceedings of INDICON 2009 - An IEEE India Council Conference. - 9781424448593
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Konferensbidrag (refereegranskat)abstract
- The onboard computer systems used in satellite launch vehicles have stringent timing requirements due the mission critical nature of their tasks. The complete control of launch vehicles is done by onboard computers (OBC) which relate to the navigation guidance, all prelaunch operations and generation of mission critical events. A fault in these systems could lead to a mission failure and catastrophic consequences. Various redundancy schemes are built into such mission critical applications to ensure the overall success of the missions. Typically a dual redundant scheme with active hot standby system has been employed in Indian Satellite launch vehicles which have been found to be very satisfactory so far. The past two decades of flight experience shows very reliable performance of the primary computers and almost no incidences demanding any serious usage of the standby computer during launches. In this paper we propose a new approach to use the standby computer to optimize the onboard computing resources which allow more slack time for further computation requirements. The slack time thus acquired can effectively be used for ‘utility enhancing computations’, such as faster control loops or specialised guidance computations. We also present a system level algorithm which depicts the overall allocation and scheduling of tasks. The proposed scheme enables an optimised usage of system resources.
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| 8. |
- Riber, Leise, et al.
(författare)
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Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome
- 2006
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 20:15, s. 2121-2134
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Tidskriftsartikel (refereegranskat)abstract
- Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.
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| 9. |
- Sharma, Atul, et al.
(författare)
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Helicobacter pylori single-stranded DNA binding protein - functional characterization and modulation of H-pylori DnaB helicase activity
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:2, s. 519-531
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.
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| 10. |
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