Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli

Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.journal articl

Virulence of functions of proteobacterial antimicrobial compound efflux (PACE) family of transport proteins

Theoretical thesis.Includes bibliographical references.1. Introduction -- 2. Material and methods -- 3. Results -- 4. Discussion -- 5. Future research -- 6. Conclusion.Acinetobacter baumannii is a significant hospital-acquired pathogen due to its traits such as multidrug resistance. Multidrug efflux systems are one of the major causes of resistance to many antimicrobials in pathogenic bacteria. The multidrug efflux pumps in bacterial pathogens also play an important role in a range of cellular activities other than drug resistance, such as virulence, biofilm formation and the export of secondary metabolites. Recent data using mouse and insect larval models suggest that the novel multidrug efflux pump AceI may play an important role in animal colonization and virulence. This project investigated the potential virulence functions of the aceI gene by performing a range of assays including growth in human serum, attachment to biotic and abiotic surfaces and iron starvation. It was found that aceI is not required for the overall fitness of the AB5075-UW for the growth in laboratory media or for the survival of this strain under iron-limiting conditions. However, an aceI knockout strain was not viable for growth in 100% human serum. Therefore, aceI might play a role in providing resistance to a component of human serum. Furthermore, biofilm formation and quorum sensing studies also suggested potential involvement of aceI in the pathophysiology of AB5075-UW.1 online resource (v, 55, xii pages : illustrations

A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

Abstract Background Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools. Results Here, we have constructed a series of all-synthetic allelic exchange vectors – pALFI1, pALFI2 and pALFI3 – with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. Conclusions We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains

Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens

Abstract The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N- and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.Peer reviewe

Systematic analyses identify modes of action of ten clinically relevant biocides and antibiotic antagonism in Acinetobacter baumannii.

Concerns exist that widespread use of antiseptic or disinfectant biocides could contribute to the emergence and spread of multidrug-resistant bacteria. To investigate this, we performed transposon-directed insertion-site sequencing (TraDIS) on the multidrug-resistant pathogen, Acinetobacter baumannii, exposed to a panel of ten structurally diverse and clinically relevant biocides. Multiple gene targets encoding cell envelope or cytoplasmic proteins involved in processes including fatty acid biogenesis, multidrug efflux, the tricarboxylic acid cycle, cell respiration and cell division, were identified to have effects on bacterial fitness upon biocide exposure, suggesting that these compounds may have intracellular targets in addition to their known effects on the cell envelope. As cell respiration genes are required for A. baumannii fitness in biocides, we confirmed that sub-inhibitory concentrations of the biocides that dissipate membrane potential can promote A. baumannii tolerance to antibiotics that act intracellularly. Our results support the concern that residual biocides might promote antibiotic resistance in pathogenic bacteria