Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
Correspondence
Susan F. Koval
skoval{at}uwo.ca
Miguel A. Valvano
mvalvano{at}uwo.ca
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ABSTRACT |
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INTRODUCTION |
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To date, the exact mechanism of B. bacteriovorus prey cell invasion and the factors contributing to prey susceptibility have not been characterized. Also, very little is known about the genetic networks regulating the developmental changes in B. bacteriovorus. Since attack-phase bdellovibrios are highly motile and cannot complete the lifecycle unless they encounter and invade prey bacteria, it is possible that motility and chemotaxis may be critical for the survival of this predator in its natural habitat. Previous studies investigating B. bacteriovorus chemotaxis in a facultative predatory strain UKi2 (now reclassified as Bacteriovorax stolpii UKi2) suggested that B. bacteriovorus is attracted to high concentrations of prey cells (Straley & Conti, 1977) and also demonstrated chemotaxis toward some amino acids (LaMarre et al., 1977
), and aerotaxis (Straley et al., 1979
). Recent work in our laboratories, using an mcp-specific oligonucleotide probe, demonstrated that at least 13 mcp (methyl-accepting chemotaxis protein) genes are present in B. bacteriovorus 109J (Flannagan, 2003
). Attack-phase bdellovibrios may benefit from a large repertoire of chemotaxis proteins to sense multiple chemical signals that would ensure efficient prey location. However, this redundancy may also complicate the interpretation of experiments where individual mcp genes are inactivated, especially if only a moderate effect on predation is observed. Lambert et al. (2003)
reported that a B. bacteriovorus mcp mutant is a less efficient predator, but in general the role of motility during Bdellovibrio predation has not been examined in detail. One of the major difficulties hampering genetic manipulations in Bdellovibrio is the prey dependence of the bacteria. Thus, it may be difficult to introduce mutations in Bdellovibrio genes directly involved in predation as well as in genes regulating the physiological transitions of the lifecycle without the potential risk of compromising the viability of the mutant cells. In this work, we used a genetic strategy to investigate the role of motility in Bdellovibrio predation by functionally disrupting the flagellar motor through the expression of a motA antisense RNA.
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METHODS |
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Determination of predation by B. bacteriovorus.
Co-cultures containing equal amounts (approx. 109 bacteria ml-1) of E. coli prey cells and B. bacteriovorus 109J were incubated in 125 ml side-arm flasks in a final volume of 20 ml HM buffer [3 mM HEPES (Sigma), pH 7·6, with 1 mM CaCl2 and 0·1 mM MgCl2] (Thomashow & Rittenberg, 1978c). The number of B. bacteriovorus 109J cells was determined microscopically as described below. Co-cultures were incubated at 30 °C with agitation, and predation was measured as a decrease in culture turbidity over time using a KlettSummerson photoelectric colorimeter with a green filter.
Molecular biology techniques.
DNA manipulations were performed by standard methods (Sambrook et al., 1989). Southern blot hybridizations were done on positively charged Biotrans nylon membranes at 60 °C in a standard hybridization buffer for digoxenin (DIG)-based probes, as described by the manufacturer (Roche Diagnostics). After hybridization the membranes were washed at room temperature in 2·0x SSC, 0·1 % SDS and at 60 °C in 0·5x SSC, 0·1 % SDS. Hybridization was detected by chemiluminescence using CSPD (Roche Diagnostics). PCR amplifications were done with a HYBAID Omnigene thermal cycler or an MJ Research PTC-200. DNA sequencing was performed at the DNA Sequencing Facility of the J. P. Robarts Research Institute (University of Western Ontario), and the sequences were examined with the program BLAST-X (http://www.ncbi.nlm.nih.gov/).
Molecular cloning of the B. bacteriovorus motAB operon and flanking genes.
Table 1 summarizes the properties of the plasmids used in this study. The plasmid pRF1 was obtained from a library of random DNA fragments from B. bacteriovorus 109J, which was specifically constructed for the cloning of mcp genes (Flannagan, 2003
). This plasmid contains three EcoRI fragments, one of which carries an mcp gene, as demonstrated by Southern blot hybridization with an mcp-specific oligonucleotide probe (Flannagan, 2003
). The DNA sequence of pRF1 also revealed an incomplete open reading frame homologous to the motility gene motB. To isolate the motAB operon from B. bacteriovorus 109J, a motB-specific probe was generated as follows. A 724 bp motB DNA fragment was amplified from pRF1 by PCR with Taq DNA polymerase (Roche Diagnostics) using the primers 5'-TAAGTCGACAAGAACATGAGAACCATGAGAGATG-3' and 5'-TTAGTCGACACTAAATTTCAACCTTGGAACTGTC-3'. The PCR product was DIG-labelled using the PCR DIG-labelling mix (Roche Diagnostics). Thermal cycling conditions for the amplification of the motB-specific probe were: 5 cycles at 95 °C for 2 min, 53 °C for 1 min, 72 °C for 1 min, followed by 25 cycles at 95 °C for 1 min, 54 °C for 45 s and 72 °C for 1 min, and a final cycle at 95 °C for 1 min, 54 °C for 45 s, and 72 °C for 5 min. This probe was used to identify genomic DNA fragments of B. bacteriovorus which were sufficiently large to carry the entire motB gene and upstream sequences (data not shown). A control experiment showed that the motB probe was specific for B. bacteriovorus as it did not hybridize to chromosomal DNA of the E. coli K-12 strain RP437. B. bacteriovorus chromosomal DNA fragments digested with ApaI, which were between 5 and 7 kb, were purified from the agarose gel with the QIAquick gel extraction kit (Qiagen). The purified DNA fragments were ligated into pBluescriptII that was also digested with ApaI. Transformants were pooled and screened by sequential Southern blot hybridization until a single plasmid hybridizing to the probe was isolated. The plasmid containing the 6·1 kb ApaI fragment was named pRF4 (Table 1
) and its DNA insert fully sequenced (GenBank accession no. AY363247; Fig. 1
).
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Construction of a motA antisense expression plasmid.
A fragment of 251 bp, encoding the first 83 amino acids of the B. bacteriovorus MotA protein (corresponding to nucleotides 42944544 of GenBank accession number AY363247) was amplified using primers 5'-ATCTAGAATGGACAAAGCAACATGGATTGGG-3' and 5'-TGAATTCCAGTCGACGATTTCGTTGAGGC-3' (EcoRI site underlined). PCR amplification was done with Pwo DNA polymerase using the following thermal cycling conditions: 95 °C for 2 min, followed by 26 cycles of 95 °C for 30 s, 54 °C for 30 s and 72 °C for 45 s, and a final extension at 72 °C for 2 min. The resulting amplicon was digested with EcoRI and ligated into pMMB206, which was digested with EcoRI and Asp700. This strategy resulted in the cloning of the 251 bp amplicon in the antisense orientation, resulting in plasmid pRF11 (Table 1). The antisense orientation of the motA DNA insert was confirmed by DNA sequencing using the sequencing primer 5'-GGCTCGTATGTTGTGTGGAATTGTG-3'.
Microscopy techniques.
For the enumeration of B. bacteriovorus, aliquots (100 µl) of B. bacteriovorus cultures were dispensed into Eppendorf tubes and stained with 2 mM 4',6-diamidino-2-phenylindole (DAPI, Molecular Probes) on ice for 15 min. A 20 µl aliquot of the stained cells was spotted onto a glass slide coated with 0·8 % agarose that had been allowed to dry. DAPI-stained bdellovibrios were visualized with a Zeiss AxioskopII microscope at a magnification of 1000x using a mercury lamp and a blue filter with an excitation wavelength of 365 nm and an emission wavelength of 420 nm. The mean number of B. bacteriovorus cells was determined by counting 10 random fields of view for each culture. The difference in the mean number of cells per field of view between each culture was corrected for by the volume of the culture added to prey cells to ensure that all cultures were inoculated with approximately the same number of bdellovibrios. Predation at various times during the co-culture of E. coli and B. bacteriovorus was visualized by phase-contrast and transmission electron microscopy. Optical images were captured using a QIMAGING Retiga 1300 cooled mono 12-bit camera and Northern Eclipse version 6.0 software from Empix Imaging. For transmission electron microscopy, 5 ml samples of co-cultures were centrifuged, resuspended in 1·5 ml fixative (2·5 % glutaraldehyde in 0·1 M sodium cacodylate buffer, pH 7·4) and left overnight at 4 °C. Cells were then fixed with 1 % (w/v) osmium tetroxide and 1 % (w/v) uranyl acetate and enrobed in agar. Samples were dehydrated in an ethanol series and embedded in LR White resin. Thin sections were cut and stained with 2 % uranyl acetate and lead citrate. Sections were examined with a Philips EM300 electron microscope operating at 60 kV.
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RESULTS |
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Gfp expression in B. bacteriovorus
The identification of the motAB genes provided us with the opportunity to directly test whether flagellar activity is indeed an essential feature in the B. bacteriovorus lifecycle. Because of the obligate nature of this predator, we sought to use a strategy based on the functional disruption of motAB genes by antisense RNA expression. B. bacteriovorus can be conjugated with plasmids belonging to the incompatibility group Q (IncQ), which replicate autonomously in this bacterium (Cotter & Thomashow, 1992a). Thus, we determined whether the IncQ plasmid pMMB206, encoding chloramphenicol resistance, could be conjugated from E. coli into B. bacteriovorus 109J. For this purpose, we first transformed E. coli ML35 prey cells with pBR325, which confers chloramphenicol resistance but has a ColE1 replicon that cannot replicate autonomously in B. bacteriovorus (Cotter & Thomashow, 1992a
) and is not transferable. Successful conjugation of pMMB206 into B. bacteriovorus was confirmed by the ability of exconjugant bdellovibrios to prey on E. coli ML35(pBR325) cells in the presence of chloramphenicol. Predation was determined by following the decrease in turbidity of the co-cultures due to the lysis of prey cells. Light microscopy of co-culture samples confirmed the lysis of prey cells and revealed an abundance of highly motile free-swimming bdellovibrios (data not shown). Prey cell lysis by B. bacteriovorus 109J(pMMB206) was delayed (occurring at 2436 h) relative to that observed in routine maintenance cultures of B. bacteriovorus 109J without the plasmid (occurring at 1618 h). To confirm that B. bacteriovorus exconjugants indeed carried pMMB206, total DNA from these bacteria was isolated and transformed into E. coli DH5
. Chloramphenicol-resistant E. coli DH5
transformants carrying pMMB206 were recovered, while no transformants containing pBR325 (that would have been derived from prey cells) were isolated.
Next, we placed the promoterless gfpmut3a gene encoding green fluorescent protein under the control of the Ptaclac promoter in pMMB206 to evaluate whether this promoter can function in B. bacteriovorus 109J. The resulting construct, pRF10, was conjugated into B. bacteriovorus 109J and the exconjugants were examined by fluorescence microscopy. Fluorescent bdellovibrios could be seen as intense localized green fluorescent patches within bdelloplasts (Fig. 2ac). Control cultures of B. bacteriovorus with the parental plasmid pMMB206 did not fluoresce. To eliminate the possibility that the localized pattern of Gfp expression observed could be due to the infection of the plasmid donor strain during conjugation, we carried out an additional control experiment where E. coli SM10(pRF10) was directly used as prey without conjugation into B. bacteriovorus. The bdelloplasts obtained in this case did not fluoresce (Fig. 2d
f). Therefore, the observation of fluorescent B. bacteriovorus cells within the bdelloplasts of prey cells demonstrated that pMMB206 was functional in this bacterium and provided a visual confirmation that a plasmid had indeed been introduced into B. bacteriovorus. However, gfp expression in B. bacteriovorus was not regulated by the inducer IPTG. The Ptaclac promoter was not altered since E. coli DH5
cells transformed with plasmid pRF10 expressed Gfp in an IPTG-dependent manner (data not shown). Therefore, it is possible that in Bdellovibrio the Ptaclac promoter is non functional, and another promoter from the plasmid backbone drives the transcription of gfpmut3a.
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DISCUSSION |
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To our knowledge, there are no studies reporting the expression of recombinant DNA in B. bacteriovorus. We constructed a derivative of pMMB206 with the gfpmut3A gene under the control of Ptaclac to demonstrate whether this promoter is functional in B. bacteriovorus. Detectable fluorescence by B. bacteriovorus exconjugants containing the reporter plasmid was observed during the growth phase within bdelloplasts, but these results occurred regardless of the presence of the inducer IPTG. Therefore, it is likely that the Ptaclac promoter in B. bacteriovorus is not functional and another constitutive promoter from the pMMB206 backbone is driving a low-level expression of gfpmut3A. These results are consistent with the possibility discussed above that plasmid-encoded genes may be preferentially expressed during the periplasmic growth phase. However, our results clearly demonstrate that pMMB206 can be used to express recombinant genes in B. bacteriovorus.
A hallmark of the B. bacteriovorus lifestyle is the transition between the presence of a polar flagellum in highly motile attack-phase cells and the loss of this structure following entry into the prey bacterium and during most of the periplasmic growth phase. The flagellum is re-formed shortly before lysis of the bdelloplast and release of progeny attack-phase bdellovibrios. The predatory lifecycle of B. bacteriovorus revolves around its ability to swim. In E. coli or Salmonella typhimurium, disruption of the motA or motB gene results in paralysis of bacterial motility despite the presence of an intact flagellum (Blair & Berg, 1988; Silverman et al., 1976
). The MotA and MotB proteins constitute the stationary part of the flagellar motor (Chun & Parkinson, 1988
; De Mot & Vanderleyden, 1994
; Garza et al., 1995
) and form a complex that facilitates the traffic of protons from the periplasmic space into the cytosol of the bacterial cell (Blair & Berg, 1990
; Braun et al., 1999
; Stolz & Berg, 1991
; Zhou et al., 1998
), which is required for flagellar rotation (Manson et al., 1977
). Thus, the B. bacteriovorus motA gene was an excellent candidate for functional gene disruption studies using antisense RNA expression. B. bacteriovorus 109J carrying plasmid pRF11, expressing motA antisense RNA, showed three abnormal phenotypes: (i) pronounced delay in the escape of progeny from the bdelloplast, (ii) tumbling swimming pattern of released attack-phase bdellovibrios, and (iii) morphological abnormalities of the bdelloplasts. None of these phenotypes were observed with B. bacteriovorus carrying the vector pMMB206. Thus, our results clearly demonstrate that the delayed lysis found with B. bacteriovorus encoding the motA antisense gene fragment was associated with an inhibition of the release of predator progeny from the bdelloplasts. The exact mechanism causing these alterations in bdellovibrios carrying pRF11 remains to be determined, but it is not likely to be due to the metabolic demand of producing a random RNA transcript, since cultures with pMMB206, which encodes the LacZ
fragment in place of the motA antisense fragment in plasmid pRF11, did not show a comparable delay in prey cell lysis. The tumbling motility of attack-phase cells could be explained by a partial defect in motility due to an incomplete inhibition of motAB expression by the antisense mRNA, which may be expressed at a low level as in the case of the Gfp protein. Although production of antisense mRNA could not be directly verified, the data support the notion that expression of the cloned motA antisense gene fragment may have downregulated the function of the motA gene.
In conclusion, we have cloned the motAB operon in B. bacteriovorus. We also show that the presence of a plasmid encoding an antisense motA gene fragment is associated with a marked delay in the escape of B. bacteriovorus progeny from bdelloplasts and with altered motility of attack-phase cells, suggesting that optimum functioning of the flagellar motor may play an important role in the Bdellovibrio lifestyle. Furthermore, antisense RNA expression strategies may become a useful genetic tool to unravel the function of other components participating in the developmental cycle of this obligate predator. Additional experiments are currently under way in our laboratories to elucidate the molecular mechanism of the motA antisense effect and to identify more suitable and efficient promoters for constitutive and regulated gene expression in B. bacteriovorus.
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ACKNOWLEDGEMENTS |
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Received 12 September 2003;
revised 1 December 2003;
accepted 12 December 2003.
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