A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.A complex network of regulatory factors governs the production of secondary metabolites and other virulence factors in the opportunistic pathogen Pseudomonas aeruginosa. This network regulates gene expression in response to stimuli such as growth phase, culture density, and oxygen and iron availability (12,26,28,37). Central components of the network are the las and rhl quorum-sensing systems, which activate gene expression in response to culture density (13). Each system is made up of two genes, one encoding an enzyme which produces a specific acylated homoserine lactone autoinducer (lasI/rhlI), and a second encoding a transcriptional activator that binds the corresponding autoinducer (lasR/rhlR). The las system directs expression of virulence factors such as elastases A and B and alkaline protease (16,25). The rhl system directs expression of rhamnolipid biosynthesis enzymes, pyocyanin biosynthesis enzymes, and hydrogen cyanide synthase (3,24,28). In addition, LasI/R regulates expression of both itself and rhlI (1, 26). The las and rhl systems together have been shown to influence the expression of over two hundred genes (36).Recently, a third signaling system based on 2-heptyl-3-hydroxy-4-quinolone, designated the Pseudomonas quinolone signal (PQS), has been shown to be a part of the quorumsensing regulatory network in P. aeruginosa (27). The production of PQS depends on lasR (27), and exogenous PQS strongly induces expression of elastase B and rhlI in a lasR mutant background (22). These results place PQS between the las and rhl quorum-sensing systems in the quorum-sensing regulatory network (22).We have described a process ("paralytic killing") in which P. aeruginosa PAO1 rapidly kills the nematode Caenorhabditis elegans by cyanide poisoning (8, 14). Previous studies of a different P....

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein ␣-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal ␣-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immunoelectron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 m/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium that is virulent towards a wide range of organisms, including bacteria, plants, nematodes, insects, and mammals (5,9,17,19,35,36,41,48,49,62). In humans, P. aeruginosa chronically infects the lungs of most cystic fibrosis patients, causes serious infections of burn wounds and eye lesions, and causes systemic infections of immunocompromised individuals (21,29,33,39). The bacterium's pathogenic versatility is reflected in its large arsenal of secreted and surface-associated virulence factors and in the complexity of the regulatory circuitry with which it controls these factors. Among the specific virulence factors that it produces are adhesins, such as pili and filamentous hemagglutinin (14, 39); protein toxins, such as phospholipase, proteases, and ADP-ribosylating enzymes (39, 64); and small-molecule poisons, such as phenazines, rhamnolipid biosurfactant, and cyanide (4,8,44). Additionally, the genome of P. aeruginosa boasts the highest proportion of predicted regulatory genes of any of the bacterial genomes sequenced to date (61), which is indicative of the bacterium's remarkable ability to adapt and thrive in numerous pathogenic and nonpathogenic environments.Several model systems for Pseudomonas pathogenesis have been developed recently, and numerous genes required for virulence towards model hosts are also required for virulence towards mammals. For example, mutants of P. aeruginosa PA-14 exhibiting reduced virulence towards Arabidopsis or Caenorhabditis elegans also exhibit reduced virulence in a burned-mouse infection model (49,50,62). In addition, a putative Pseudomonas signal transduction gene cluster requi...

Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCEAcinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.A cinetobacter baumannii is a Gram-negative opportunistic pathogen that causes infections with serious morbidity and mortality and is one of a group of six pathogens responsible for most multidrug-resistant (MDR) nosocomial infections (the ESKAPE pathogens, i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (1, 2). The pathogen is infamous for its ability to persist in hospital settings, a feature that reflects its capacity for long-term survival on abiotic surfaces through resistance to desiccation and disinfectants (3).Genomic and molecular epidemiological studies of A. baumannii isolates have helped define the pathogen's global population structure, its antibiotic resistance gene repertoire, the size and content of its pangenome, and phylogenetic relationships among outbreak strains (3-6). Three primary clonal lineages (GC1 to GC3) appear responsible for the majority of hospital outbreaks globally (7). Although these lineages display restricted genetic diversity among core genes (7), the species' genome is actually quite dynamic. Strains display striking variability in accessory gene content (5, 8), including antibiotic resistance genes (9), even among related isolates of a single outbreak (10). This genomic variability presumably reflects the actions of transmissible plasmids, insertion elements, phage, integrons, natural transformation, and reco...

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is a category A select agent. We created a sequence-defined, near-saturation transposon mutant library of F. tularensis novicida, a subspecies that causes a tularemia-like disease in rodents. The library consists of 16,508 unique insertions, an average of >9 insertions per gene, which is a coverage nearly twice that of the greatest previously achieved for any bacterial species. Insertions were recovered in 84% (1,490) of the predicted genes. To achieve high coverage, it was necessary to construct transposons carrying an endogenous Francisella promoter to drive expression of antibiotic resistance. An analysis of genes lacking (or with few) insertions identified nearly 400 candidate essential genes, most of which are likely to be required for growth on rich medium and which represent potential therapeutic targets. To facilitate genome-scale screening using the mutant collection, we assembled a sublibrary made up of two purified mutants per gene. The library provides a resource for virtually complete identification of genes involved in virulence and other nonessential processes.essential genes ͉ promoter ͉ tularemia ͉ U112

Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

Twenty-five fetuses with limb body wall complex (LBW complex) were evaluated. The diagnosis was based on two out of three of the following: exencephaly/encephalocele with facial clefts; thoraco- and/or abdominoschisis; and limb defect. Ninety-five percent (24/25) of the fetuses had associated internal structural defects. In 72% (18/25) the internal defects have been recognized as being secondary to vascular disruption. Concordance was not found between the side and location of the body wall defect versus the limb, internal, and cranial defects. In 85% there was evidence for persistence of the extraembryonic coelom by examination of the placenta. In this same group (85%) there was persistence of the ectodermal-amnion margin, with the amnion being continuous with the skin of the body wall defect. In 40% (10/25) there were tags and amniotic adhesions at other sites. There was no difference in the types or incidence of internal defects between those with and those without amniotic bands. The abnormalities in this collection and experimental animal models support vascular disruption during 4-6 weeks' gestation as an etiology for LBW complex. There is disruption and loss of existing tissues, persistence of embryonic structures, and secondary malformations. Persistence of the extraembryonic coelom may lead to the typical amniotic tags, ring constrictions, and adhesions seen in some specimens.

We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle method), a method which can be used with virtually any transposon. The procedure reliably identified more than 100,000 transposon insertions in a single experiment, providing near-saturation coverage of the genome. To test the effectiveness of the procedure for mutant identification, we screened for mutations reducing intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic tobramycin resistance had been previously analyzed at genome scale using mutant-by-mutant screening and thus provided a benchmark for evaluating the new method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the majority of which were then verified with individual mutants. The group of genes with the strongest mutant phenotypes included nearly all (13 of 14) of those with strong mutant phenotypes identified in the previous screening, as well as a nearly equal number of new genes. The results thus show the effectiveness of the Tn-seq method in defining the genetic basis of a complex resistance trait of P. aeruginosa and indicate that it can be used to analyze a variety of growth-related processes.