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. 2010 Aug 25:10:225.
doi: 10.1186/1471-2180-10-225.

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

Carla Cacciotto  1 , Maria Filippa Addis, Daniela Pagnozzi, Bernardo Chessa, Elisabetta Coradduzza, Laura Carcangiu, Sergio Uzzau, Alberto Alberti, Marco Pittau
Affiliations

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

Carla Cacciotto et al. BMC Microbiol. .

Abstract

Background: Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.

Results: The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.

Conclusions: This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

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Figures

Figure 1
Figure 1
Total protein patterns and Western immunoblotting reactivity of M. agalactiae PG2T proteins. Panel A. Coomassie blue staining. Panel B: Immunoblotting reactivity obtained with antibodies against the P48 lipoprotein. From left to right: M: molecular weight standards in kDa; T: total protein pattern; H: hydrosoluble protein fraction; L: liposoluble protein fraction obtained after Triton X-114 fractionation
Figure 2
Figure 2
2-D PAGE patterns of M. agalactiae PG2T protein extracts. Left: 2-D PAGE of a M. agalactiae PG2T total protein extract. Right: 2-D PAGE of M. agalactiae PG2T liposoluble proteins obtained after Triton X-114 fractionation.
Figure 3
Figure 3
2-D PAGE map of M. agalactiae PG2T liposoluble proteins illustrating protein identifications obtained by mass spectrometry. Proteins are indicated by grouping all individual identifications corresponding to the same protein in a series of spots.
Figure 4
Figure 4
2D DIGE of liposoluble proteins extracted from M. agalactiae PG2T and two field strains. Overlay image: image generated from the superimposition of the signals generated by the three samples. White indicates presence of the protein spot in all three isolates. Panels A, B, and C represent isolates PG2T, Nurri, and Bortigali, respectively. Panels D, E, and F represent the superimposition of Nurri/Bortigali, PG2T/Nurri, and PG2T/Bortigali, respectively.
Figure 5
Figure 5
GO graph of proteins identified by 2-D PAGE-MS and GeLC-MS/MS in the Triton X-114 fraction of M. agalactiae PG2T. Protein identifications are classified according to cellular localization.
Figure 6
Figure 6
GO graph of proteins identified by GeLC-MS/MS in the Triton X-114 fraction of M. agalactiae PG2T. Protein identifications are classified according to function
See this image and copyright information in PMC

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