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Objectives: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants., Results: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His 6 -tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate., Conclusions: Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Objectives: To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene., Results: NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His 6 -tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O 2 -insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate., Conclusions: NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.

Neisseria meningitidis is a gram-negative bacterium that lives as a commensal in the human nasopharynx. Meningococci are generally non-invasive, but can invade the nasopharyngeal epithelia and enter the bloodstream causing life-threatening illnesses. It is generally thought that meningococci do not survive for long outside the host, and that transmission requires relatively close contact between hosts. There are some reports, however, that meningococci can survive drying on surfaces, including glass, plastic and cloth. Our examination of N. meningitidis strains dried on glass showed differences in survival of isolates belonging to serogroups B, C and W135, including persistence of Cuban, New Zealand, and Norwegian epidemic strains up to 8 days, depending on temperature and humidity. Survival of a New Zealand epidemic strain isolate NZ98/254 under ambient conditions in the laboratory was greatest in winter suggesting that environmental factors impacted survival. For most isolates, including NZ98/254, survival under controlled conditions at 30 °C was greater at 22% than 30% relative humidity. There were also some differences in survival between carriage and invasive strains. The results suggest that N. meningitidis could be transmitted through contact with surfaces outside the host, potentially including contact through shared drinking vessels.

Neisseria meningitidis (meningococcus) can cause meningococcal disease, a rapidly progressing and often fatal disease that can occur in previously healthy children. Meningococci are found in healthy carriers, where they reside in the nasopharynx as commensals. While carriage is relatively common, invasive disease, associated with hypervirulent strains, is a comparatively rare event. The basis of increased virulence in some strains is not well understood. New Zealand suffered a protracted meningococcal disease epidemic, from 1991 to 2008. During this time, a household carriage study was carried out in Auckland: household contacts of index meningococcal disease patients were swabbed for isolation of carriage strains. In many households, healthy carriers harbored strains identical, as determined by laboratory typing, to the ones infecting the associated patient. We carried out more-detailed analyses of carriage and disease isolates from a select number of households. We found that isolates, although indistinguishable by laboratory typing methods and likely closely related, had many differences. We identified multiple genome variants and transcriptional differences between isolates. These studies enabled the identification of two new phase-variable genes. We also found that several carriage strains had lost their type IV pili and that this loss correlated with reduced tumor necrosis factor alpha (TNF-α) expression when cultured with epithelial cells. While nonpiliated meningococcal isolates have been previously found in carriage strains, this is the first evidence of an association between type IV pili from meningococci and a proinflammatory epithelial response. We also identified potentially important metabolic differences between carriage and disease isolates, including the sulfate assimilation pathway. IMPORTANCE Neisseria meningitidis causes meningococcal disease but is frequently carried in the throats of healthy individuals; the factors that determine whether invasive disease develops are not completely understood. We carried out detailed studies of isolates, collected from patients and their household contacts, to identify differences between commensal throat isolates and those that caused invasive disease. Though isolates were identical by laboratory typing methods, we uncovered many differences in their genomes, in gene expression, and in their interactions with host cells. In particular, we found that several carriage isolates had lost their type IV pili, a surprising finding since pili are often described as essential for colonization. However, loss of type IV pili correlated with reduced secretion of a proinflammatory cytokine, TNF-α, when meningococci were cocultured with human bronchial epithelial cells; hence, the loss of pili could provide an advantage to meningococci, by resulting in a dampened localized host immune response.

Summer Gut Syndrome (SGS) is caused by various Vibrio bacterial species and can have negative effects on aquaculture farms worldwide. In New Zealand, SGS is caused by Vibrio harveyii infecting King Salmon (Oncorhynchus tshawytscha). To find leads for the prevention of SGS, we screened the inhibitory effects of 16 strains of Shewanella upon V. harveyii growth in competitive solid phase cultures. The detailed investigation of Shewanella halifaxensis IRL548 revealed 2-benzyl-4-chlorophenol (1), a known, commercially available antibacterial agent, as the major bioactive component. Synthesis of a small library of congeners to confirm the natural product identity and to provide a structure-activity relationship for the observed activity was also completed. Compound 1 exhibits moderate activity against two pathogenic microorganisms., (Copyright © 2016. Published by Elsevier Ltd.)

Neisseria meningitidis is the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen, Neisseria meningitidis is frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using an in vitro assay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 A resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 A). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Delta cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Delta cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Delta cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Bartonella bacteria adhere to erythrocytes and persistently infect the mammalian bloodstream. We previously identified four highly conserved Bartonella quintana adhesin genes that undergo phase variation during prolonged bloodstream infection. The variably expressed outer membrane proteins (Vomp) encoded by these genes are members of the trimeric autotransporter adhesin family. Each B. quintana Vomp appears to contribute a different adhesion phenotype, likely mediated by the major variable region at the adhesive tip of each Vomp. Although studies document that the Vomp adhesins confer virulence phenotypes in vitro, little is known about in vivo virulence strategies of Bartonella. We sought to determine whether the B. quintana Vomp adhesins are necessary for infection in vivo by using a vomp null mutant. It first was necessary to develop a system to generate in-frame deletions of defined genes by allelic exchange in a wild-type Bartonella background, which had not been achieved previously. We utilized sacB negative selection to generate a targeted, in-frame, markerless deletion of the entire vomp locus in B. quintana. We also recently developed the first animal model for B. quintana infection, and using this model, we demonstrate here that the deletion of the entire vomp locus, but not the deletion of two vomp genes, results in a null mutant strain that is incapable of establishing bloodstream infection in vivo. The Vomp adhesins therefore represent critical virulence factors in vivo, warranting further study. Finally, our allelic exchange strategy provides an important advance in the genetic manipulation of all Bartonella species and, combined with the animal model that recapitulates human disease, will facilitate pathogenesis studies of B. quintana.

Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms.

A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccS (diminished capacity to colonize; Cj1223c-Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A DeltadccR response regulator mutant generated using the virulent strain 81-176 background exhibited significantly reduced colonization of immunocompetent limited flora (I-LF) mice, severe combined immunodeficient limited flora (SCID-LF) mice, and 1-day-old chicks. A DeltadccS sensor kinase mutant was likewise defective for colonization in the I-LF mouse model. DeltadccR-infected SCID-LF mice also exhibited dramatically reduced inflammation relative to wild type-infected SCID-LF mice. Despite this diminished colonization capacity, the DeltadccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.

The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods [corrected].

The ordered assembly of the Caulobacter crescentus flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarchy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.

Lagenidium giganteum, a facultative parasite of mosquito larvae, cannot synthesize sterols, and requires an exogenous source of these lipids in order to enter its reproductive cycle. This parasite grows vegetatively in the absence of sterols, but requires cholesterol or structurally related compounds to produce motile zoospores, which are the only stage capable of infecting mosquitoes. Sterols structurally related to cholesterol and some steryl esters inhibited the activity of L. giganteum phospholipase A2 (PLA2), an enzyme that hydrolyzes fatty acids from the sn-2 position of glycerophospholipids. Sterols that induce reproduction inhibited L. giganteum PLA2 activity, while sterols and steroids that do not support sporulation had minimal effect. Most steryl esters had no effect on enzyme activity, but cholesteryl arachidonate (CA) was a potent inhibitor of parasite PLA2. Not all enzymes partly purified using a DEAE-Sephacel column were affected by these lipids, demonstrating selective inhibition of specific enzymes. Potency was enhanced by up to several orders of magnitude if epoxy fatty acids were esterified to the cholesterol nucleus. The steryl ester pool was dynamic during morphogenesis, and the fatty acid composition of the steryl esters did not mimic total cell or membrane (glycerophospholipid) fatty acid composition as L. giganteum proceeded through its growth cycle. Synthesis of CA and monoepoxy CA by the parasite was confirmed using electrospray mass spectrometry and collision-induced dissociation. Steryl derivatives selectively inhibited PLA2 enzymes from bovine pancreas, snake venom, and human cytoplasmic 85-kDa PLA2.

The mosquito pathogenic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) is a sterol auxotroph that can grow vegetatively in the absence of these compounds, but requires an exogenous source of sterols to enter its sexual and asexual reproductive cycles. Electrospray mass spectrometry (MS) and electrospray MS/MS were used to examine three major glycerophospholipid molecular species--glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE) and glycerophosphoinositol (GPI)--from fungal mycelium and nuclei grown in defined medium with and without isoprenoids which induce (cholesterol and ergosterol) or do not induce (squalene, cholestane) reproduction. Testosterone supplementation of defined media inhibited growth of L. giganteum, so the effect of this steroid on phospholipid metabolism could not be assessed. Mycelium grown in defined media supplemented with these isoprenoids produced significantly different quantities of total phospholipid relative to unsupplemented media and to each other, ranging from a mean of 292 micrograms phosphate per g wet weight for cholesterol-supplemented media to 56 micrograms phosphate per g wet weight for mycelium grown in the presence of squalene. A very large percentage of the GPC (69-80 mol%) and GPI (74-79 mol%) molecular species from mycelia and nuclei contained ether linkages. GPE molecular species had 13-20 mol% ether-containing moieties. The elevated levels of ether lipids may be related to the sterol auxotrophic nature of the fungus. Isoprenoid supplementation of defined growth media resulted in many significant changes in molecular species for all three lipid classes. Significant differences (P < 0.05) in the percentage of total cell ether lipids in GPC and GPE were generated by isoprenoid supplements to culture media. Mycelium grown in the presence of the two sterols which induce asexual and sexual reproduction in L. giganteum, cholesterol and ergosterol, had a significantly greater percentage of ether-containing GPE moieties. The glycerolipid species from nuclei isolated from cultures grown with cholesterol and ergosterol were similar to the composition of nuclei isolated from fungus cultured in defined medium without any supplement or supplemented with squalene. The nuclear membrane from mycelia grown in cholestane-supplemented media, however, had a very different glycerophospholipid composition relative to either whole cells or nuclei from cells grown on other media. It appears that one of the reasons that cyclic isoprenoids such as cholestane do not induce fungal reproduction is that they drastically alter the nuclear membrane glycerophospholipid composition.