Rf O'Toole

Tetracycline-inducible gene regulation in mycobacteria.

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non‐motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500‐fold decrease in virulence when fish were inoculated by immersion in bacteria‐containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon‐insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in‐frame deletion was constructed in the coding region of this gene. The resulting non‐chemotactic mutant exhibited wild‐type pathogenicity when injected intraperitoneally into fish but showed a decrease in virulence similar to that seen for the non‐motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum

Expression of zebrafish cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation.

Cxcl8 is a pro-inflammatory chemokine, best known for its role in neutrophil chemotaxis. Signalling through its receptors, Cxcr1 and Cxcr2, is induced by inflammatory stimuli evoked by microbial, chemical or environmental stress, and hormonal signals. While it is recognised that Cxcl8 signalling is active in the gut mucosa, this is not as well understood as its role in leukocyte trafficking. Here, we report the characterisation of genes encoding the zebrafish Cxcl8, Cxcr1 and Cxcr2. By a combination of genomic, expression and functional analyses, we show that the Cxcl8 signalling pathway is conserved in zebrafish. As in humans, cxcl8 is expressed in zebrafish leukocytes. Transcripts were also detected in intestinal epithelial cells, and this expression is upregulated under inflammatory conditions caused by bacterial or chemical insult. Expression of cxcr1 and cxcr2 is robust within the developing gut. This work provides a model for the study of Cxcl8 signalling during gut inflammation.

A Two-Component Regulator of Universal Stress Protein Expression and Adaptation to Oxygen Starvation in Mycobacterium smegmatis

We identified a response regulator in Mycobacterium smegmatis which plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c of M. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. A devR mutant of M. smegmatis was adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in the devR mutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream of devR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the alpha-crystallin (HspX) orthologue of M. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of the hspX promoter to gfp demonstrated that DevR directs gene expression when M. smegmatis enters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, the devR mutant was 10(4)-fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress in M. smegmatis.

Role of Motility in Adherence to and Invasion of a Fish Cell Line by Vibrio anguillarum

To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum, both in vivo and in vitro.

Universal stress proteins and Mycobacterium tuberculosis

Mycobacterium tuberculosis expresses universal stress proteins (USPs) when its growth is retarded by oxygen depletion. This class of proteins is emerging as being important in the resistance of bacteria to stress and prolonged growth arrest. Here we assess the properties of USPs and their relevance to mycobacteria.

RpoN of the fish pathogen Vibrio (Listonella) anguillarum is essential for flagellum production and virulence by the water-borne but not intraperitoneal route of inoculation

To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative sigma 54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.

Comparison of the microalgal community within fast ice at two sites along the Ross Sea coast, Antarctica

Diverse microbial communities survive within the sea ice matrix and are integral to the energy base of the Southern Ocean. Here we describe initial findings of a four season survey (between 1999–2004) of community structure and biomass of microalgae within the sea ice and in the underlying water column at Cape Evans and Cape Hallett, in the Ross Sea, Antarctica as part of the Latitudinal Gradient Project. At Cape Evans, bottom-ice chlorophyll a levels ranged from 4.4 to 173 mg Chl a m−2. Dominant species were Nitzschia stellata, N. lecointei, and Entomoneis kjellmanii, while the proportion of Berkeleya adeliensis increased steadily during spring. Despite being obtained later in the season, the Cape Hallett data show considerably lower standing stocks of chlorophyll ranging from 0.11 to 36.8 mg Chl a m−2. This difference was attributed to a strong current, which may have ablated much of the bottom ice biomass and provided biomass to the water below. This loss of algae from the bottom of the ice may explain why the ice community contributed only 2% of the standing stock in the total water column. Dominant species at Cape Hallett were Nitzschia stellata, Fragilariopsis curta and Cylindrotheca closterium. The low biomass at Cape Hallett and the prevalence of smaller-celled diatoms in the bottom ice community indicate that the ice here is more typical of pack ice than fast ice. Further data will allow us to quantify and model the extent to which ice-driven dynamics control the structure and function of the sea ice ecosystem and to assess its resilience to changing sea ice conditions.

Experimental models used to study human tuberculosis.

Mycobacterium tuberculosis causes more deaths in humans than any other bacterial pathogen. The most recent data from the World Health Organization reveal that over 9million new cases of tuberculosis occur each year and that the incidence appears to be increasing with population growth. Despite the global burden of tuberculosis, we are still reliant on relatively dated measures to prevent, diagnose, and treat the disease. New, more effective tools are needed to diminish the incidence of tuberculosis. M. tuberculosis lacks a natural host beyond humans and, hence, surrogate models have been employed in the study of the pathogen. The discovery and development of new vaccines, diagnostics, or antitubercular drugs are dependent upon the validity of any experimental model used and its relevance to tuberculosis in humans. In this review, a range of experimental models, from in vitro studies with fast-growing low-pathogenic species of mycobacteria to the infection of nonhuman primates with virulent M. tuberculosis, will be discussed.