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1.
Figure 1

Figure 1. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Growth rate of the M. fortuitum strains 10851/03, 10860/03 and DSM 46621. The growth rate of the strains was measured by quantification of the ATP-content [displayed as relative light units (RLU)] in broth cultures.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
2.
Figure 3

Figure 3. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Occurrence of porin genes in M. fortuitum. Chromosomal DNA of different strains was digested with SacII and analysed by Southern Blotting using a probe derived from the porM1 sequence. Lane 1: M. fortuitum 10851/03; lane 2: M. fortuitum 10860/03; lane 3: M. fortuitum DSM 46621.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
3.
Figure 7

Figure 7. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Effect of down-regulation and over-expression of porM1 and porM2 on the growth of M. fortuitum. M. fortuitum strains 10851/03 (A-E) and DSM 46621 (F-N) were electoprorated with plasmids pSHKLx1 (A, F), pSRr106 (B, G), pMV261 (C, H, L), pSRb101 (D, I, M) or pSRb103 (E, K, N), plated on either 100 μg ml-1 hygromycin (A, B), 100 μg ml-1 kanamycin (C-K) or 25 μg ml-1 kanamycin (L-N) and incubated for four days.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
4.
Figure 6

Figure 6. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Complementation of the porin-deficient mutant strain M. smegmatis ML10 with porM1 and porM2. M. smegmatis ML10 was transformed with the control vector pMV306 (A), the mspA-containing plasmid pSSa100 (B), the porM1-containing plasmid pSRa102 (C) and the porM2-containing plasmid pSRa104 (D). After electroporation of the plasmids, dilutions of the transformed bacteria were plated onto Mycobacteria 7H11 agar with 25 μg ml-1 kanamycin and incubated for four days. Panel (E) illustrates the result of an independent experiment showing the time course of the appearance of the colonies on Mycobacteria 7H11 agar with 25 μg ml-1 kanamycin during four days after plating of a 1:10 dilution of the electroporated cells.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
5.
Figure 4

Figure 4. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Alignment of PorM1 and PorM2 from M. fortuitum and MspA and MspC from M. smegmatis. The start codon ATG and the stop codon TGA were chosen according to the sequence of mspA. The cleavage recognition site of the signal peptidase was predicted for PorM1, PorM2 and MspC using the SignalP 3.0 Server at http://www.cbs.dtu.dk/services/SignalP/[11]. The predicted signal peptide cleavage sites corresponded to the signal peptide cleavage site of MspA [6]. Identical amino acids are dark grey, similar amino acids are light grey and different amino acids are not shaded. For PorM1 and MspA an identity index of 94.8% was calculated, while PorM2 showed an amino acid identity of 90.7% to MspA.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
6.
Figure 5

Figure 5. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Detection of PorMs in M. fortuitum and M. smegmatis. 2D-Electrophoresis, Western Blot, ELISA and qRT-PCR experiments proved PorMs to be expressed in the analysed strains. Section A shows 2D-Electrophoresis of protein isolation from the strain M. fortuitum 10860/03 using the detergent nOPOE. The arrow indicates the porin spot proven by Western Blot analysis (see Additional file 2). Section B and C show comparative analysis of porin expression among RGM. Expression of porin was detected by means of ELISA (B) and qRT-PCR (C). Each value represents the mean (± SD) of at least three independent experiments. B: Quantification of porin by means of ELISA in cell extracts of different mycobacteria using the polyclonal antibody pAk MspA#813. C: RT-Real-time-PCR quantification of porin mRNA in various RGM using specific primers and probes for mspA or porM, respectively.

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.
7.
Figure 2

Figure 2. From: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth.

Map of genomic regions containing porM1 from M. fortuitum 10860/03 and porM2 from M. fortuitum 10851/03. Section A shows a 2895 bp region representing the insert of plasmid pSSp107. The insert includes the porM1 gene and three other ORFs. Up- and downstream to porM1 various nucleotide signal sequences were detected: -10 signal of a promoter (TATGTT), a ribosome binding site (RBS: GGAGA), a signal peptide recognition sequence (SP) of 81 bp and a hairpin structure, which could represent a terminator. Furthermore, the location of the antisense fragment selected for the generation of plasmid pSRr106 is indicated. Section B represents a 1697 bp region of M. fortuitum 10851/03 containing porM2 and two other ORFs. Upstream to porM2 a -10 signal of a promoter (TACGTT), a ribosome binding site (AGGGAGAA) and a signal peptide recognition sequence (SP) of 93 bp were identified. Subsequences were predicted using the software packages MacVector™ 7.2.3 (Accelrys) and Lasergene (DNASTAR).

Soroush Sharbati, et al. BMC Microbiol. 2009;9:31-31.

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