Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

Deletion mutants and animal models have been instrumental in the study of Helicobacter pylori pathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement during H. pylori colonization and chronic infection. To achieve this goal, we adapted the Escherichia coli Tn10-derived tetracycline-inducible expression system for use in H. pylori. The ureA promoter was modified by inserting one or two tet operators to generate tetracycline-responsive promoters, named uPtetO, and these promoters were then fused to the reporter gfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboring tetR and uPtetO-GFP was characterized by measuring GFP activity and by immunoblotting. The two tet-responsive uPtetO promoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of the uPtetO-GFP construct and the nature of the promoter driving expression of tetR influenced the strength of the uPtetO promoters upon induction. Integration of uPtetO-GFP and tetR constructs at different genomic loci was stable in vivo and did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expression in vivo during chronic infection. These results open new experimental avenues for dissecting H. pylori pathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.     

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.     

Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.     

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer.     

AHR,a novel acute hypoxia‐response sequence,drives reporter gene expression under hypoxia in vitro and in vivo

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter‐driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia‐response sequence)‐GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP‐expressing vector under the control of CMV (cytomegalovirus promoter‐GFP). We transduced AHR‐GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV‐GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR‐GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR‐GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia‐activated gene expression system.     

Oxidative Stress Response and Characterization of the oxyR-ahpC and furA-katG Loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H₂O₂, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

Integrative elements for Bacillus subtilis yielding tetracycline-dependent growth phenotypes

We describe the construction and application of elements for random insertion of promoter containing DNA into the genome of Bacillus subtilis. The outward-facing promoter of these integrative elements termed InsTet^G+ is inducible by tetracycline so that conditional mutants are generated. We constructed three InsTet^G+ variants using different regulatory windows. In the first, the regulator gene tetR is located within the element, allowing one-step mutagenesis. The second contains tetR in the chromosome and yields the best regulation efficiency. The third exploits xylose-dependent tetR expression from a plasmid, enabling induction of TetR synthesis so that distinct expression levels of an affected gene can be adjusted. We have obtained mutant strains with all three variants. For some of them, growth can be modulated by the presence of effectors. Most growth defects occur in the presence of inducers, presumably due to regulated expression of antisense RNA.     

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.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .     

Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells

Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-gamma. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-gamma-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-gamma-induced expression of HLA-DRalpha and HLA-DRbeta mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRalpha and HLA-DRbeta mRNA in response to IFN-gamma. Histone acetylation induced by IFN-gamma at the HLA-DRalpha promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRalpha promoter in M. avium-infected THP-1 cells stimulated with IFN-gamma. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.     

The application of lucifferase as a reporter of environmental regulation of gene expression in mycobacteria

This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

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.     

天蓝色链霉菌孢子形成的关键基因——whiG的诱导表达

与链霉菌分化有关的whiG结构基因已亚克隆到链霉菌表达载体pAK203的tipA启动子下游.该启动子能被硫链丝菌素诱导.whiG基因的诱导表达不仅可使天蓝色链霉菌孢子形成缺陷突变株C71恢复产生孢子的能力,而且也能使天蓝色链霉菌野生型菌株J1501和变铅青链霉菌TK54的孢子形成更为丰满.Western杂交也进一步证实了诱导表达后whiG基因产物——σ~(whiG)产量的增加.这为依赖于σ~(whiG)RNA多聚酶的发育调控启动子体外转录的研究提供了有利条件.