Microarray profiling of host-extract-induced genes and characterization of the type VI secretion cluster in the potato pathogen Pectobacterium atrosepticum

Abstract

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.

Regulation of Type VI Secretion Gene Clusters by σ 54 and Cognate Enhancer Binding Proteins

“…One alternate hypothesis is that bEBP phosphorylation occurs through a quorum-sensingdependent signaling pathway or is dependent upon a sensor kinase and a specific environmental signal which remain to be identified. Interestingly, the Pectobacterium atrosepticum T6SS gene cluster has been shown to be induced by cell extracts of potato tubers (31,32), suggesting that the bacteria sense specific compounds and provide an output response leading to T6SS expression. In all these cases, defining the interplay or convergence of different regulatory pathways is essential to an understanding of the complete network of regulation.…”

“…Among them, type VI secretion systems (T6SS) were first identified in 2006 in a screen for identifying bacterial factors necessary for Vibrio cholerae resistance to amoeba predation (40). Since then, T6SS have been characterized or described for many Gram-negative bacteria, including animal and human pathogens (Pseudomonas aeruginosa, Burkholderia species, pathogenic Escherichia coli, Francisella tularensis, Salmonella enterica), plant pathogens (Pectobacterium atrosepticum, Pseudomonas syringae, Agrobacterium tumefaciens), and fish pathogens (Edwardsiella tarda) as well as for soil, marine, and environmental bacteria (2,15,32,36,38,42,45,48). They are thus widely distributed in a broad spectrum of bacteria in a wide range of environmental niches, but they are also involved in a broad diversity of processes (6,12,17,41,46,47).…”

“…P-Labeled probes were obtained by PCR amplification of the promoter sequences from chromosomal DNA using dNTP mixes containing [␣- 32 P]deoxyadenosine triphosphate. Labeled probes were column purified (Wizard SV gel and PCR cleanup kit; Promega) to remove radioactive nucleotides.…”

Nooks and Crannies in Type VI Secretion Regulation

“…In Pectobacterium atrosepticum, the T6SS gene cluster is induced by the presence of potato tuber extracts (69,70), whereas the Agrobacterium tumefaciens T6SS is induced under acidic conditions in the presence of low concentrations of phosphate, conditions found in proximity to wounded plant cells (115). Similarly, the T6SS gene cluster of the plant pathogen Pseudomonas fluorescens is induced by the presence of necrotic roots (7).…”

Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

“…Samples of induced proteins were picked for matrix-assisted laser desorption ionization-time of flight (TOF) mass spectrometry (MS) and quadrupole TOF MS. Protein identification and data analysis were performed in a manner similar to that previously described (28).…”