Introduction

A chemolithotrophic microorganism species that can utilize reduced sulfur compounds as a substrate for growth, was isolated from halophilic and alkaliphilic environments and classified as belonging to the genus Thialkalivibrio by Sorokin et al. (2001). A member of this genus, Thialkalivibrio versutus is a Gram-negative, halophilic, alkaliphilic, sulfur-oxidizing and chemolithotrophic bacterium. This bacterium derives its energy by oxidizing reduced or partially reduced sulfur compounds and also fixes CO2 from the atmosphere. Thialkalivibrio is of interest for exploring the treatment of industrial waste water from sulfide (Klok et al. 2012; van den Bosch et al. 2008). Moreover, T. versutus is preferred as a model haloalkaliphilic organism owing to its ability to grow well in defined medium with reduced sulfur compounds as substrates (Banciu et al. 2004).

The physiology of T. versutus has been well studied. So far, however, no methods have been developed to introduce foreign genes into this extremely haloalkaliphilic bacterium. Conjugation is an important method of horizontal gene transfer in bacteria. The genetic material is transferred from one bacterium to another via direct cell-to-cell contact. Liu et al. (2007) developed a conjugative transfer system between E. coli and the moderately thermophilic, extremely acidophilic bacterium, Acidithiobacillus caldus MTH-04. Tan et al. (2014) transformed plasmids into target bacteria via conjugative transfer to engineer Halomonas TD01. Conjugation was originally a robust method of introducing foreign genes into recipient cells before the development of transformation methods by heat shock or electroporation.

In this study, we have developed a set of conjugation transfer systems for Thialkalivibrio and subsequently also devised an effective expression system based on the broad-host-range plasmid pBBRMCS1. The derived plasmids can be transferred into host cells by conjugation. High-efficiency promoters were selected based on the observed expression levels of red fluorescent protein (RFP) from a reporter construct. Subsequently, a plasmid carrying the Vitreoscilla hemoglobin (VHb) gene (vgb) under the control of a highly efficient tac promoter was transformed into SOB306 strain and the effect of the expression of this gene was further evaluated. To our knowledge, this is the first time that a plasmid transfer method and expression system have been developed for Thialkalivibrio strains, which will lay the foundation for further studies at the genetic level.

Materials and methods

Strains and medium

The bacterial strains and plasmids used in this study are listed in Supplementary Table 1. Thialkalivibrio versutus SOB306 is a mutant strain of T. versutus D301. The recipes of TD and MTD media are described in Supplementary Table 2. E. coli strains were cultured in lysogeny broth at 37 °C and 220 rpm. T. versutus SOB306 strains grew in a defined medium with thiosulfate (TD) at 30 °C and 180 rpm. The solid medium was made by adding 1.5% agar in liquid media.

Conjugation transfer

Conjugation between T. versutus SOB306 and E. coli was performed on MTD solid medium by filter mating. The donor and recipient cells were harvested by centrifugation at 7000×g for 10 min when the OD600 reached 0.6–0.7. Both donor and recipient cells were washed twice or thrice with MTD liquid medium without yeast extract, resuspended in the same solution, and mixed at a ratio of 1:1 (v/v). One hundred microliters of this mixture were then transferred to filter membranes (0.45 µm pore size, 50 mm diameter) which were placed on MTD solid medium plates. The conjugation plates were cultured at 37 °C for 6, 12 18 and 24 h. The cultures were washed from the membrane with 5 ml washing liquid mentioned above and plated on streptomycin selective and nonselective TD solid media. The donor and recipient cells were also plated on respective selective and non-selective media as controls. The selection plates containing the exconjugants and the T. versutus SOB306 parent were incubated at 30 °C for 5 days while plates containing the donor E. coli were incubated at 37 °C for 24 h. Finally, colonies recovered in the experimental groups were counted and the transfer frequency was calculated using the following formula:

$$ {\text{TF}}_{\text{r}} = \frac{{{\text{N}}_{\text{S}} }}{{{\text{N}}_{\text{T}} }} \times 100\% , $$
(1)

where TFr represents the transfer frequency, NS represents the number of colonies on the selective medium, and NT represents the number of colonies on the nonselective medium.

Primers used in this study are listed in Supplementary Table 2.

Observation of RFP fluorescence and analysis of fluorescence intensity

Fluorescence of cells was observed at 550 nm (excitation). The fluorescence intensity of different cells was analyzed by Spectramax M5, Molecular Devices. The relative fluorescence intensity was calculated by actual fluorescence intensity of bacterial liquids dividing by the OD600 value.

The characterization of VHb and determination of sulfur compounds

The expression of gene was detected by CO difference spectra at 419 nm (Liao et al. 2014). Concentration of sulfate and thiosulfate were determined by an ICS-900 ion chromatograph system (Xu et al. 2015).

Results and discussion

Conjugative transfer between E. coli and T. versutus SOB306

To assess the effect of conjugation time, the donor and recipient cells were mixed and plated on MTD solid medium at 6, 12, 18 and 24 h, respectively. The exconjugants were then washed out and plated on solid TD medium containing streptomycin. After 5 days, all plates had colonies, with the greatest number of colonies on the plate corresponding to 6 h conjugation (Supplementary Fig. 1). To confirm the transfer of the two plasmids into T. versutus SOB306, colony PCR was performed by primers HX-smF/R and M13F/R. Transfer frequency of pBBR-smr was less than that of pKT230 within the same contact time (Supplementary Table 3). Compared to E. coli sm10, E. coli s17 had a greater ability to act as donor to transfer broad-range-host plasmids into recipient cells. The results were caused due to the diversity of physiological characteristics between the two donor cells. Finally, the stability of plasmids pBBR-smr and pKT230 in T. versutus SOB306 was tested. After five passages, about 59% SOB306 cells still carried pKT230, but only 43% cells retained pBBR-smr (Supplementary Table 3). These results indicated that pKT230 was more stable than pBBR-smr in T. versutus SOB306.

We constructed an expression system for T. versutus SOB306 based on broad-host-range plasmid pBBR1MCS owing to its small size and abundant multiple cloning sites. A few high-scoring promoters were selected based on the prediction results of online software mentioned in Supplementary Table 4 (Reese 2001). The efficiency of these promoters was then tested by reporter constructs. Positive colonies were selected for resistance to chloramphenicol and then screened for resistance to streptomycin. We noted that the cells did not show resistance to streptomycin after modifying the construct. Eight promoters were inserted into pBBR-sm and then transformed in SOB306 strain by conjugative transfer. Of the eight clones obtained, those carrying four promoters (pBBR-sm-p3/6/32/tac) regained resistance to streptomycin, while the other four were still sensitive. We verified that these promoters could promote transcription of the downstream strAB gene. RNA polymerase from E. coli may have poor affinity with some promoters of T. versutus SOB306, which is a likely explanation as to why pBBR-sm-p1/2/4/5 clones were not resistant to streptomycin in E. coli. To test the strength of these promoters, we employed another plasmid, pBBR-rfp. Like the previous constructs in pBBR-sm, all promoters were cloned into KpnI/HindIII sites of pBBR-rfp and transformed into DH5α competent cells.

We could visually identify expression of rfp only in cells carrying pBBR-rfp-p3 and pBBR-rfp-ptac, which developed cherry red color (Supplementary Fig. 2). Cells carrying three clones (pBBR-rfp-p1/p5/p32) were bright cherry red only when concentrated by centrifugation. Additionally, SOB306 carrying pBBR-rfp-p3 and pBBR-rfp-ptac clones were cherry red with an intensity similar to those in E. coli. Fluorescence intensity of strains carrying rfp gene under the control of p3 and tac promoters was checked to evaluate the effectiveness of these promoters. As shown in Table 1, p3 and ptac can effectively promote the expression of rfp gene. In E. coli, the fluorescence intensities of s17(pBBR-rfp-p3) and s17(pBBR-rfp-ptac) were 234 and 635 times that of wild type s17, respectively. The values in corresponding T. versutus SOB306 were 281 and 982 times, respectively. Therefore, we concluded that tac was more effective than p3 in both bacteria.

Table 1 Fluorescence intensity of cell with different promoters
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Expression of Vgb in T. versutus SOB306

Wakabayashi et al. (1986) reported that a Gram negative bacterium, Vitreoscilla, expressed a hemoglobin (VHb) encoded by Vitreoscilla globin gene (vgb). VHb can bind O2 and deliver it to the cell respiratory chain, thus enhancing respiration under hypoxic conditions and facilitating cell growth and accumulation of metabolic products (Geckil et al. 2001).

Plasmid pBBR-vgb-ptac was transferred into T. versutus SOB306 by conjugation. CO difference spectrum assay was used to determine expression of VHb in T. versutus SOB306. CO binds to the expressed bioactive VHb and generates a CO–VHb chromophore complex. This complex can be detected at ~419 nm. The non-recombinant SOB306 strain was used as a negative control in the assay. The results showed that the CO-binding complex appeared at ~419 nm (Fig. 1). This indicated that vgb was expressed in T. versutus SOB306 and had potent CO-binding activity.

Fig. 1
figure 1

CO-difference spectra of T. versutus SOB306-vgb+ for check vgb gene expression

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Growth of T. versutus SOB306 expressing VHb and its desulfurization activity

Wild type and recombinant SOB306 strains were cultured in TD medium in a shake-flask under aerobic conditions. Growth curves were derived based on viable cell number and OD600 values (Fig. 2). As shown in Fig. 2a, OD values of both strains were almost the same during the initial growth phase. However, the curve of recombinant SOB306 showed a steep rise from 25 h. Finally, OD of the recombinant strain reached the highest OD value of 16 ± 0.75 at 40 h and 7.6 ± 0.86 at 48 h (the end of culture). By contrast, the highest value of OD of wild type was only 2.41 ± 0.11. The viable cell density of the recombinant strain was close to that of the wild strain during the entire growth phase (Fig. 2b). SOB306 carrying and expressing vgb did not have an obvious growth advantage over the wild type strain. Furthermore, the expression of vgb gene appeared to cause a slight inhibition of cell growth, which is similar to that observed in E. coli (Geckil et al. 2001) and Ganoderma lucidum (Li et al. 2016). The highest value of OD of a vgb+ strain is approx. 6.7 times of that of a vgb− strain. The growth conditions of the two strains for different culture times are shown in Supplementary Fig. 3. Liquid transmission of light containing of T. versutus-vgb+ was weaker than that of T. versutus-vgb−. This may be attributed to hemoglobin mediated changing metabolism of cells by enhanced transport of oxygen into the cellular respiratory chain (Yang et al. 2015).

Fig. 2
figure 2

Comparison of growth of OD600 of cultures of T. versutus SOB306-vgb+ and wild type (vgb−) under aerobic conditions, a OD600 values and b cell numbers

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The concentrations of thiosulfate and sulfate were measured to determine the change of sulfur metabolism of these two kinds of strains. As shows in Fig. 3a, the vgb+ strain can metabolize thiosulfate faster than the vgb− strain. Thialkalivibrio species mainly derive energy from the oxidization of low-valence sulfur compounds. The rate of thiosulfate oxidation is closely related to cell growth. Moreover, thiosulfate was removed completely by the vgb+ strain late in growth, but 4% of thiosulfate was not utilized by the vgb− strain in the same phase of growth. The average rate of removal of thiosulfate was increased from 43 ± 1.6 to 55 ± 1.9% owing to VHb expression during the entire growth period. Therefore, the vgb+ strain had higher desulfurization activity when compared to that of the vgb− strain owing to its better performance in utilization of thiosulfate. As in case of thiosulfate oxidation, the formation of sulfate in the vgb+ strain is also faster than that in the vgb− strain (Fig. 3b). Xiong et al. reported that the expression of vgb gene in Rhodococcus erythropolis LSSE8-1 markedly improved its desulfurizing activity (Xiong et al. 2007).

Fig. 3
figure 3

Comparison of desulfurizing activity of T. versutus SOB306-vgb+ and wild type (vgb−) under aerobic conditions, a the consumption and removal rate of thiosulfate and b production of sulfate

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The supply of O2 is very important for cell growth and product formation in aerobic microorganisms. Although the cell density of the SOB306 strain was not very high owing to its chemolithotrophic properties, a large number of sulfur globules produced by the strain could potentially hinder O2 transfer. Therefore, VHb could help SOB306 utilize the dissolved O2 more efficiently.

Conclusion

A highly efficient genetic transformation system based on conjugative transfer method in the genus Thialkalivibrio was developed. A broad-range-host plasmid pBBRMCS1 was used to select strong promoters for SOB306 strain. Two promoters, tac (generally used in E. coli) and p3, regulated transcription of peroxiredoxin gene in SOB306 strain and promoted expression of the rfp gene in SOB306. The strength of the tac promoter was about three times of p3. Plasmid pBBR-tac-vgb expressing VHb was then constructed and transformed into SOB306 through conjugation transfer and this boosted oxidation of thiosulfate as well as cell metabolism. However, cell growth was slightly decreased, which is different from results reported earlier. To our knowledge, this is the first time that a genetic transformation strategy and expression system have been built in the genus Thialkalivibrio. It lays a foundation to boost research into metabolic engineering and helps to develop potentially high activity strains for biodesulfurization.