Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide

Seven different recombinant bioluminescent strains of Escherichia coli containing, respectively, the promoters katG and soxS (responsive to oxidative damage), recA (DNA damage), fabA (membrane damage), grpE, and rpoE (protein damage) and lac (constitutive expression) fused to the bacterial operon from Photorhabdus luminescens, were used to describe the mechanism of toxicity of 1,1-dimethylhydrazine (1,1-DMH) on bacteria, as well as to determine whether bacteria can sensitively detect the presence of this compound. A clear response to 1,1-DMH was observed only in E. coli carrying the katG’::lux, soxS’::lux, and recA’::lux-containing constructs. Preliminary treatment with catalase of the medium containing 1,1-DMH completely diminished the stress-response of the P_katG, P_recA, and P_soxS promoters. In the strain E. coli (pXen7), which contains a constitutive promoter, the level of cellular toxicity caused by the addition of 1,1-DMH was dramatically reduced in the presence of catalase.It is suggested that the action of 1,1-DMH on bacterial cells is determined by hydrogen peroxide, which is formed in response to reduction of the air oxygen level.     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

Tn5/7-lux: a versatile tool for the identification and capture of promoters in Gram-negative bacteria

Background

The combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.

Results

We devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir⁺ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.

Conclusions

Although mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.     

GroEL chaperone binding to beetle luciferases and the implications for refolding when co-expressed

The folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.     

Improved lux reporters for use in Staphylococcus aureus

The use of luxABCDE (lux) offers certain advantages over other reporters, such as: lacZ and xylE. It is real time and its signal generation is produced without the requirement for any additional substrates. In some bacteria such as Staphylococcus spp, light production by luciferase is restricted because of a limited availability of endogenous substrates such as fatty acid aldehyde. We describe the construction of promoterless-lux cloning vectors, pGYlux and pAmilux. S. aureus carrying B. subtilis xyl/tetO promoter fused to the lux genes of pGYlux gave up to a 2.5-fold enhancement of luminescence over S. aureus carrying the xyl/tetO promoter fused to lux genes of the previously published parent vector pAL2. Furthermore, pAmilux showed a 6-fold enhancement of lux expression when compared to pGYlux in S. aureus. This was achieved by cloning the constitutive ami promoter upstream of the luxCDE genes to increase endogenous fatty acid aldehyde production while maintaining its reporter functionality by fusing promoters to the luxAB genes.     

Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation

Bioluminescent bacterial sensors are based upon the fusion of bacterial bioluminescence (lux) genes, acting as a reporter element, to selected bacterial stress-response gene promoters. Depending upon the nature of the promoter, the resulting constructs react to diverse types of environmental stress, including the presence of toxic chemicals, by dose-dependant light emission. Two bacterial sensors, harbouring sulA::luxCDABE and grpE::luxCDABE fusions, activated by the model chemicals nalidixic acid (NA) and ethanol, respectively, were subjected to molecular manipulations of the promoter region, in order to enhance the intensity and speed of their response and lower their detection thresholds. By manipulating the length of the promoter-containing segment (both promoters), by introducing random or specific mutations in the promoter sequence or by duplicating the promoter sequence (sulA only), major improvements in sensor performance were obtained. Improvements included significantly enhanced sensitivity, earlier response times and an increase in signal intensity. The general approaches described herein may be of general applicability for optimizing bacterial sensor performance, regardless of the sensing or reporting elements employed.     

Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

The Effects of Regulatory Proteins RcsA and RcsB on the Expression of the Vibrio fischeri lux Operon in Escherichia coli

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA–RcsB activated lux expression and, consequently, the bioluminescence of E. coli cells in the early log phase.     

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for real‐time, low‐light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Such luciferin–luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea panzy (Renilla reniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein (GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expression experiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and to visualize the proliferation of tumour cells in animal models. Copyright © 2002 John Wiley & Sons, Ltd.     

Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora

The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions −578 to −501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coliσ ⁷⁰ promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28° C, relative to expression at 37° C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora. Received: 26 February 1997 / Accepted: 23 May 1997     

The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.     

Development of a Plasmid Vector for Easy Selection of Strong Promoters

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of β-lactamase activity in liquid or solid media. Received: 26 July 1999 / Accepted: 22 November 1999     

Isolation of active DNA-binding nuclear proteins from tomato galls induced by root-knot nematodes

We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsiveLEMMI9 and 35S promoters. In noninfected tissue, the method based on nuclei enrichment through a Percoll cushion was superior for isolation of DNA-protein binding activity with both promoters. With infected roots; the method based on crude extracts performed better with theLEMMI9 promoter, whereas nuclei-enriched extracts worked better with the 35S promoter. Therefore, both methods can be used to extract proteins for DNA-binding assays from infected roots, but the method of choice may depend on the promoter under study. Both authors have contributed equally to this work.     

Organization of the lux genes of photobacterium phosphoreum

The lux genes from Photobacterium phosphoreum (NCMB844) have been cloned into Escherichia coli in a plasmid containing the T7-bacteriophage promoter. By specific expression in vivo under the T7 promoter, five structural genes (luxA-E) coding for the fatty acid reductase and luciferase polypeptides were identified as well as a new gene, designated as luxF, which codes for a 26kDa polypeptide. This new gene is located between luxB and luxE and thus disrupts the structural gene order of luxCDABE found in the Vibrio genus. The luxF gene and the protein it codes for have recently been identified in other Photobacterium species and so appears to be widely distributed within this genus. Nucleotide sequencing of the luxF gene has shown it to code for a protein homologous to the luciferase subunits, coded by the luxA and luxB genes. Although this gene is not necessary for light emission in all luminescent bacteria, it must play an essential role in the biochemistry, physiology, or ecology of the luminescent system in species of the Photobacterium genus.