Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitor

Quantitative image analysis for the characterization of microbial aggregates in biological wastewater treatment : a review

Quantitative image analysis techniques have gained an undeniable role in several fields of research during the last decade. In the field of biological wastewater treatment (WWT) processes, several computer applications have been developed for monitoring microbial entities, either as individual cells or in different types of aggregates. New descriptors have been defined that are more reliable, objective, and useful than the subjective and time-consuming parameters classically used to monitor biological WWT processes. Examples of this application include the objective prediction of filamentous bulking, known to be one of the most problematic phenomena occurring in activated sludge technology. It also demonstrated its usefulness in classifying protozoa and metazoa populations. In high-rate anaerobic processes, based on granular sludge, aggregation times and fragmentation phenomena could be detected during critical events, e.g., toxic and organic overloads. Currently, the major efforts and needs are in the development of quantitative image analysis techniques focusing on its application coupled with stained samples, either by classical or fluorescent-based techniques. The use of quantitative morphological parameters in process control and online applications is also being investigated. This work reviews the major advances of quantitative image analysis applied to biological WWT processes.The authors acknowledge the financial support to the project PTDC/EBB-EBI/103147/2008 and the grant SFRH/BPD/48962/2008 provided by Fundacao para a Ciencia e Tecnologia (Portugal)

Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in <i>Salmonella</i> Typhimurium

<div><p>In this study we adapted a Mu<i>d</i>-based delivery system to construct a random <i>yfp</i> reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of <i>Salmonella</i> Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational <i>yfp</i> insertion in <i>iscR</i> and <i>iolR</i>, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.</p></div

A multifaceted study of Pseudomonas aeruginosa shutdown by virulent podovirus LUZ19

&lt;p&gt;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. IMPORTANCE: 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&lt;/p&gt;</p

Strains, phages and plasmids used in this study.

<p>Strains, phages and plasmids used in this study.</p

Merged phase contrast and YFP images of growing LT2 <i>iscR</i>::<i>yfp</i> cells (previously grown overnight at 40–45 MPa and 37°C) after pressure release, showing (A) typical cells with a bilobed nucleoid, and (B–F) occasionally occurring cells with a “trilobed” nucleoid (indicated by arrows).

<p>Scale bar corresponds to 5 μm.</p

Construction and use of the MudY transposon.

<p>(A) During construction of the Mu<i>d</i>Y transposon, the <i>lacZYA</i>-<i>npt</i> of Mu<i>d</i>K was replaced by an <i>yfp</i>-<i>frt</i>-<i>cat</i>-<i>frt</i> module through recombineering (blue and green crosses indicate the homologous regions involved in recombination). (B) The <i>frt</i>-<i>cat</i>-<i>frt</i> cassette can be readily flipped out using the Flp recombinase, thereby reducing possible polar effects of the <i>cat</i> marker. (C) Exact location and genomic context of two Mu<i>d</i>Y insertions (<i>iscR</i>::Mu<i>d</i>Y and <i>iolR</i>::Mu<i>d</i>Y) in LT2 yielding endogeneous nucleoid reporters.</p

Representative images showing the colocalization of the IscR144::YFP and IolR159::YFP proteins with the DAPI stained nucleoid in LT2 <i>iscR</i>::Mu<i>d</i>Y (A–D) and LT2 and <i>iolR</i>::Mu<i>d</i>Y (E–H), respectively.

<p>Phase-contrast (A,E), DAPI (B,F), YFP (C,G), and merged (D,H) images are shown. Inset in panel C includes a larger image of an LT2 <i>iscR</i>::Mu<i>d</i>Y cell showing the apparent helical shape of the nucleoid highlighted by IscR144::YFP. Scale bars correspond to 5 μm.</p