On-chip microbial culture for the specific detection of very low levels of bacteria.

Microbial culture continues to be the most common protocol for bacterial detection and identification in medicine and agronomics. Using this process may take days to identify a specific pathogen for most bacterial strains. Surface Plasmon Resonance (SPR) detection is an emerging alternative technology that can be used for the detection of bacteria using protein microarrays although typical limits of detection are in the range of 10(3)-10(6) cfu mL(-1), which is not compatible with most Food Safety regulation requirements. In this work, we combine concomitant "on-chip" microbial culture with sensitive SPR detection of bacteria thus allowing rapid specific detection of bacteria pathogens - including Salmonella enterica serovar Enteritidis, Streptococcus pneumoniae and Escherichia coli O157:H7 - cultured on a protein microarray. This Culture-Capture-Measure (CCM) approach significantly decreases both the number of processing steps and the overall assay time for bacterial detection. Signal analysis of SPR responses allowed the fast and quantitative assessment of bacterial concentrations initially present in the sample as low as 2.8 ± 19.6 cfu per milliliter. Altogether, our results show how simple, easy-to-operate, fluidic-less and lo-tec microarrays can be used with unprocessed samples and yield - in a single assay - both qualitative and quantitative information regarding bacterial contamination.

Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane.

Pseudomonas aeruginosa is a Gram-negative bacterium that can inhabit a wide variety of environments and infect different hosts. Human infections by this microbe are nowadays perceived as a major health problem due notably to its multiresistance. The present data set provides the first description of P. aeruginosa inner membrane proteome. To achieve this, we combined efficient separation of membranes from PAO1 reference strain using discontinuous sucrose gradient centrifugation and MS-based proteomic analysis. A core list of 991 nonredundant proteins was established and analyzed in terms of trans-membrane domains, signal peptide, and lipobox sequence prediction. Furthermore, functional insights into membrane-spanning and membrane-associated protein complexes have been explored. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000107.

An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.