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In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection.
Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus.
[Show abstract][Hide abstract] ABSTRACT: A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks.
[Show abstract][Hide abstract] ABSTRACT: Giant bacteriophages phi KZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phi KZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The 4 KZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201 phi 2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.
[Show abstract][Hide abstract] ABSTRACT: Giant bacteriophages phiKZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phiKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The phiKZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201phi2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.
[Show abstract][Hide abstract] ABSTRACT: Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these - often uncharacterized - proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32x10(6) independent clones), was screened against early proteins (gp1 to 9) of phiKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage-host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to phiKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the bacteriophage proteins expressed immediately after infection of the host cell. Most of these early proteins are probably involved in bacteriophage-host interactions redirecting the bacterial metabolism to phage production. Interaction analysis of the first 16 phiKMV gene products (gp) identified homotypic interactions of gp7, gp9 and gp15. Two related yeast two-hybrid procedures, a matrix and a minilibrary approach, were applied to detect protein-protein interactions. A two-step selection procedure enabled drastic reduction of the background. Interactions were confirmed by drop tests. Multimerization of gp15 is consistent with its putative function as a DNA helicase involved in DNA replication. Homotypic interaction of gp7 and gp9 suggests they function as dimers or multimers. The absence of heterotypic interactions among early phiKMV proteins hints at their functional independence from other early phage proteins and their involvement in phage-host interactions that are important for creating optimal conditions for phage propagation. Besides, these results demonstrate the compatibility of phiKMV early gene products with the yeast two-hybrid system. Therefore, they are promising candidates to screen for interactions with host proteins.
Archives of Virology 02/2007; 152(8):1467-75. DOI:10.1007/s00705-007-0967-4 · 2.39 Impact Factor