Sebastian Poggio

Molecular Mechanisms of Ethanol-Induced Pathogenesis Revealed by RNA-Sequencing

Acinetobacter baumannii is a common pathogen whose recent resistance to drugs has emerged as a major health problem. Ethanol has been found to increase the virulence of A. baumannii in Dictyostelium discoideum and Caenorhabditis elegans models of infection. To better understand the causes of this effect, we examined the transcriptional profile of A. baumannii grown in the presence or absence of ethanol using RNA-Seq. Using the Illumina/Solexa platform, a total of 43,453,960 reads (35 nt) were obtained, of which 3,596,474 mapped uniquely to the genome. Our analysis revealed that ethanol induces the expression of 49 genes that belong to different functional categories. A strong induction was observed for genes encoding metabolic enzymes, indicating that ethanol is efficiently assimilated. In addition, we detected the induction of genes encoding stress proteins, including upsA, hsp90, groEL and lon as well as permeases, efflux pumps and a secreted phospholipase C. In stationary phase, ethanol strongly induced several genes involved with iron assimilation and a high-affinity phosphate transport system, indicating that A. baumannii makes a better use of the iron and phosphate resources in the medium when ethanol is used as a carbon source. To evaluate the role of phospholipase C (Plc1) in virulence, we generated and analyzed a deletion mutant for plc1. This strain exhibits a modest, but reproducible, reduction in the cytotoxic effect caused by A. baumannii on epithelial cells, suggesting that phospholipase C is important for virulence. Overall, our results indicate the power of applying RNA-Seq to identify key modulators of bacterial pathogenesis. We suggest that the effect of ethanol on the virulence of A. baumannii is multifactorial and includes a general stress response and other specific components such as phospholipase C.

MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.

The reducible complexity of a mitochondrial molecular machine

Molecular machines drive essential biological processes, with the component parts of these machines each contributing a partial function or structural element. Mitochondria are organelles of eukaryotic cells, and depend for their biogenesis on a set of molecular machines for protein transport. How these molecular machines evolved is a fundamental question. Mitochondria were derived from an α-proteobacterial endosymbiont, and we identified in α-proteobacteria the component parts of a mitochondrial protein transport machine. In bacteria, the components are found in the inner membrane, topologically equivalent to the mitochondrial proteins. Although the bacterial proteins function in simple assemblies, relatively little mutation would be required to convert them to function as a protein transport machine. This analysis of protein transport provides a blueprint for the evolution of cellular machinery in general.

A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides

Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a gamma-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other alpha-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster.

A Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus

Mitochondria are organelles derived from an intracellular α-proteobacterium. The biogenesis of mitochondria relies on the assembly of β-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an α-proteobacterium, and the BAM (β-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A ∼150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

A protein critical for cell constriction in the Gram-negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan-binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.

The flagellar hierarchy of Rhodobacter sphaeroides is controlled by the concerted action of two enhancer‐binding proteins

The expression of the bacterial flagellar genes follows a hierarchical pattern. In Rhodobacter sphaeroides the flagellar genes encoding the hook and basal body proteins are expressed from σ54‐dependent promoters. This type of promoters is always regulated by transcriptional activators that belong to the family of the enhancer‐binding proteins (EBPs). We searched for possible EBPs in the genome of R. sphaeroides and mutagenized two open reading frames (ORFs) (fleQ and fleT), which are in the vicinity of flagellar genes. The resulting mutants were non‐motile and could only be complemented by the wild‐type copy of the mutagenized gene. Transcriptional fusions showed that all the flagellar σ54‐dependent promoters with exception of fleTp, required both transcriptional activators for their expression. Interestingly, transcription of the fleT operon is only dependent on FleQ, and FleT has a negative effect. Both activators were capable of hydrolysing ATP, and were capable of promoting transcription from the flagellar promoters at some extent. Electrophoretic mobility shift assays suggest that only FleQ interacts with DNA whereas FleT improves binding of FleQ to DNA. A four‐tiered flagellar transcriptional hierarchy and a regulatory mechanism based on the intracellular concentration of both activators and differential enhancer affinities are proposed.

The four different σ54 factors of Rhodobacter sphaeroides are not functionally interchangeable

The σ54 factor is highly conserved in a large number of bacterial species. From the complete genome sequence of Rhodobacter sphaeroides, it was possible to identify four different sequences encoding potentially functional σ54 factors. In this work, we provide evidence that one of these copies (rpoN2) is specifically required to express the flagellar genes in this bacterium. A mutant strain carrying a lesion in the rpoN2 gene was unable to swim even though the RpoN1 and RpoN3 proteins were present in the cytoplasm. The possibility that the different copies of the σ54 factor might be specific for the transcription of a particular subset of σ54 promoters was reinforced by the fact that a mutant strain carrying a lesion in rpoN1 showed a severe growth defect in nitrogen‐free culture medium, even though the rpoN2 and rpoN4 genes were actively transcribed from a plasmid or from the chromosome. Different mech‐anisms that might be responsible for this specificity are discussed.

The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.

The β‐barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α‐, β‐, γ‐, δ‐ and ε‐proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α‐proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α‐proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ‐proteobacteria such as E. coli.

The Flagellar Protein FliL Is Essential for Swimming in Rhodobacter sphaeroides

In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.