Mutagenesis of RpoE-like sigma factor genes in Bdellovibrio reveals differential control of groEL and two groES genes
© Lambert et al.; licensee BioMed Central Ltd. 2012
Received: 9 May 2012
Accepted: 7 June 2012
Published: 7 June 2012
Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, preys upon and leaves other bacteria, or grows axenically without prey. In addition to “housekeeping” sigma factors, its genome encodes several alternate sigma factors, including 2 Group IV-RpoE-like proteins, which may be involved in the complex regulation of its predatory lifestyle.
We find that one sigma factor gene, bd3314, cannot be deleted from Bdellovibrio in either predatory or prey-independent growth states, and is therefore possibly essential, likely being an alternate sigma 70. Deletion of one of two Group IV-like sigma factor genes, bd0881, affects flagellar gene regulation and results in less efficient predation, although not due to motility changes; deletion of the second, bd0743, showed that it normally represses chaperone gene expression and intriguingly we find an alternative groES gene is expressed at timepoints in the predatory cycle where intensive protein synthesis at Bdellovibrio septation, prior to prey lysis, will be occurring.
We have taken the first step in understanding how alternate sigma factors regulate different processes in the predatory lifecycle of Bdellovibrio and discovered that alternate chaperones regulated by one of them are expressed at different stages of the lifecycle.
Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, digests, and leaves other Gram-negative bacteria, or when it grows axenically without prey [1–3]. Discrete waves of enzymes digesting different prey contents are required so that predatory enzymes do not act on each other, as the Bdellovibrio changes from a non-replicating “attack-phase” outside the prey, to a growing and replicating state inside prey. The B. bacteriovorus HD100 genome encodes several potential sigma factors for RNA polymerase which may contribute to such organised waves of gene regulation . The Bdellovibrio bacteriovorus HD100 genome has several predicted “housekeeping” sigma factors: gene bd0242 encoding an RpoD sigma 70 sigma factor; gene bd3318, encoding a FliA-like sigma factor and gene bd0843 encoding an RpoN-like sigma factor. In addition, there are two homologues of genes predicted to encode Group IV-RpoE-like sigma factors, bd0881 (product predicted at 162 amino-acids) and bd0743 (product predicted at 206 amino-acids). Further, gene bd3314 is predicted to encode a larger sigma factor homologue (predicted at 373 amino-acids) with sigma 70 homology.
RpoE-like sigma factors in other bacteria mediate gene expression in response to changes in host/external environment and bacteria with mutations in rpoEs can be defective in virulence or other host interactions . Bd0881 and Bd0743 predicted proteins show significant homology (28.6% and 31.8% identity respectively) to the rpoE gene product of E. coli which encodes a sigma factor of the ECF type that is responsive to extra-cytoplasmic, periplasmic events; RpoE in E. coli is sequestered at the inner membrane by an RseA RseB pair of proteins, until inducing-events, in the shape of abnormally folded proteins in the periplasm, cause it to be released and active . The Bdellovibrio genome, like that of other delta-proteobacteria, does not contain rseAB genes, suggesting that the RpoE-like sigma factors encoded by bd0881 and bd0743 belong more generally to the Group IV-type sigma factors. Unlike some members of this group, the Bdellovibrio genes lack the typical downstream co-transcribed gene encoding a product with homology to an anti-sigma factor. Indeed the genes (bd0745 and bd0882) that are immediately downstream of bd0743 and bd0881 are unique to the Bdellovibrio genome, with no other significant homologues in other bacteria.
We hypothesised that the regulatory functions of alternate Group IV sigma factors might be diverse and important in the Bdellovibrio lifestyle, where prey-interaction versus prey-independent axenic growth brings with it many different challenges to the cell, including outer membrane insults, and a need for a great deal of de novo protein synthesis. Thus we used directed mutagenesis with kanamycin cartridge insertion, to test if inactivation of the three sigma factor genes bd3314, bd0881 and bd0743, affected viability and to determine what their regulatory roles in the Bdellovibrio axenic and predatory lifestyles may be. We find that one is likely essential, one is involved in regulating predatory processes and one is involved in repression of different components of the GroESEL chaperone complex, which themselves may have different roles in the predatory lifecycle.
Transcriptional studies and bioinformatics show the operon structures for bd0743 and bd0881
amino acid composition of −35 recognition sites of the Bdellovibrio sigma factor gene products compared to E. coli RpoE
-35 recognition site amino acids in E. coli RpoE
Corresponding amino acid in Bd0743
Corresponding amino acid in Bd0881
Inactivation of sigma factor genes suggests that bd3314 may be essential
Kanamycin resistant cassettes were inserted into the bd0743 bd0881 and bd3314 genes to disrupt their coding sequences, and knockout mutants were screened for as described previously . Viable knock-out mutants capable of predation were obtained for bd0743 and bd0881, but they could not be obtained for bd3314, despite extensive screening in axenic (prey-independent) and predatory conditions well beyond the bacterial numbers from which the other two mutants were isolated, suggesting that Bd3314 may be essential (a total of 287 isolates were screened from 4 separate conjugation experiments, yielding only bd3314 merodiploids, compared to 10 and 29 isolates yielding 6 and 1 knock-out mutants for bd0743 and bd0881 respectively). Bd3314 is larger than the other RpoE-like sigma factors (predicted 373 amino acids compared to 162 and 206) with homology to regions 1.2, 2, 3 and 4 of sigma 70 and so this may be acting as an alternative sigma 70 factor guiding the transcription of housekeeping genes which would explain why generating a knock-out mutant was not obtained. Top hits from a BLAST search for Bd3314 are sigma-70 genes from many delta-proteobacteria, (outwith the predatory Bdellovibrio) further supporting its possible role as an alternative sigma 70 protein. Some hits from BLAST were annotated as RpoH, but Bd3314 is unlikely to be RpoH as it lacks the “RpoH box” conserved in these proteins . Further studies on the groups of genes it regulates is beyond the scope of this manuscript, but it is likely that as Bd3314 is conserved in other delta-proteobacteria, including many non-predatory bacteria, it may not have a specialised predatorily associated function.
Luminescent prey assay shows less efficient predation by a Bdellovibrio bd0881 knockout strain
To look for any evidence of association between RpoE-like sigma factor proteins and motility gene expression, we firstly measured the transcription of the 3 motA genes in ΔBd0881 and ΔBd0743, but found no difference compared to wild type (data not shown). This led us to conclude that Bd0881 does not act at motor regulation and does not produce faster rotating but shorter flagella.
Our results showed that expression of bd0881 was all but abolished at 45 min to 1 hour after Bdellovibrio addition to prey, and resumed later in the predatory cycle, before prey lysis, as shown in Figure 4 alongside expression of the critical fliC3 gene. The expression of the fliC3 gene initially drops early in the predatory cycle, then resumes as the Bdellovibrio are nearing septation and flagella are synthesised prior to prey lysis and progeny escape from the prey cell debris into liquid cultures. Thus the similarity in expression patterns of fliC3 gene and bd0881 during predation may imply that Bd0881 protein is involved in regulatory events to do with the timepoints where flagella are being synthesised, i.e. around septation, but the fact that ΔBd0881 mutants are not immotile shows that Bd0881 is not required for the “all or nothing” induction of the fliC3 gene expression itself.
RT-PCR reveals regulation of chaperone genes by Bd0743
RT-PCR was used to study the expression of GroE chaperone protein genes in wild-type and sigma-factor knockout Bdellovibrio strains, as chaperone genes are typically RpoE-regulated in other bacteria, although no obvious E. coli RpoE- like consensus sequence was seen upstream of them in the B. bacteriovorus HD100 genome. Other bacteria induce expression of GroE protein chaperones upon heat shock (typically experimentally 42°C) in order to deal with misfolded proteins . Furthermore, over-expression of chaperones can aid the expression of high levels of proteins in cells  including situations where addition of phage–encoded GroES proteins modify the size of protein that the bacterial chaperone can fold, to assemble large phage capsid proteins . The Bdellovibrio genome has, in addition to the bd0097 bd0099 groES groEL genes, a second homologue, bd3349, of groES (here designated groES2 versus groES1 for bd0097), so we investigated the expression of all these genes by RT-PCR using matched amounts of RNA from wild-type and sigma-factor mutant Bdellovibrio, treated in attack phase, at different temperatures (29°C and heat-shock 42°C for 10 mins; Figure 3) using methods previously described . In wild-type Bdellovibrio, as is the case in many other bacteria, groES1EL expression was low at normal Bdellovibrio growth temperature (29°C) and expression was induced at a higher level under heat shock (42°C). This situation was the same for wild type and the ΔBd0881 mutant indicating that the Bd0881 sigma factor is not involved in this heat shock event. In the ΔBd0743 mutant, however, groES1EL expression was de-repressed, even in non-heat shock conditions suggesting that the Bd0743 sigma factor controls, directly or indirectly, the repression of groES1EL under normal temperature conditions. The viability of the ΔBd0743 cells was not affected under predatory growth conditions as determined by plaque assay indicating that this GroE deregulation does not severely affect the cells during laboratory culturing.
The second chaperone gene groES2 (bd3349) was expressed at a very low level, in attack phase cells of in the wild-type and ΔBd0881 mutant, under both normal and heat shock conditions,(Figure 3); suggesting that possibly it is not normally part of the heat shock response and may have a different role outside. In the ΔBd0743 mutant, however, groES2 expression was de-repressed in both normal and heat shock conditions, again implying that this sigma factor controls the expression of repressors of chaperone gene expression.
We have shown that of three B. bacteriovorus HD100 sigma factor genes with at least partial rpoE homology, one- bd3314, is likely essential for Bdellovibrio cell life and cannot be deleted. bd0881 and bd0743 can be deleted with the Bdellovibrio retaining the ability to grow predatorily or prey-independently.
In the case of ΔBd0881 the predatory efficiency was reduced, despite the flagellar motility of the mutant being slightly increased, (despite a slight but statistically significant shortening of flagellar filament length) thus the change in predation efficiency may not be due to motility changes but regulation of other predatory genes. The bd0881 gene has an expression pattern across the predatory cycle that is similar to that of the flagellin genes whose expression is required for Bdellovibrio motility. That bd0881 expression is turned off and then resumes at a similar time to flagellin gene expression, during the predatory cycle, implies that Bd0881 may have a role associated with pre-septation developmental maturation of Bdellovibrio around the time that flagella are being built in newly dividing cells. However the Bd0881 sigma factor does not directly regulate the expression of fliC flagellin or mot flagellar motor genes themselves.
Surprisingly, predatory efficiency was not affected in our cultures by the slower swimming speed of the ΔBd0743 sigma factor mutant; this is probably indicative of sufficient mixing of predator and prey at close quarters in lab conditions. The slight increase in flagellar length in ΔBd0743 mutants is likely to have come with the incorporation of a higher percentage of a less rigid flagellin in the flagella causing a less efficient “bow wave” and this may account for the slower swimming. In both the ΔBd0743 and ΔBd0881 mutants, small but significant changes in swimming speed were paradoxically associated with changes apparently in the wrong direction in flagellar length. This shows that it is not simply flagellar length that governs the thrust produced by flagellar propellers. In previous studies on the six different flagellins that are incorporated into the flagellar propeller of Bdellovibrio[11, 17], we found that different flagellin compositions of a single Bdellovibrio flagellum are possible, and that in the case of a fliC4 mutant, for example, an apparently wild type-length flagellum gave a lower swimming speed than wild type  suggesting an altered filament rigidity. As flagellar filament growth, in a bacterium with six flagellins, is a post-transcriptionally highly controlled process involving diverse chaperones and gate keepers at the base of the flagellum allowing different subunits to be added into the growing flagellum  we cannot expect to tell anything meaningful about these small changes of swimming speed from simple studies of flagellar filament gene expression, so we have decided to leave this aspect of the investigation at this point.
In looking at chaperonin expression regulation by B. bacteriovorus HD100 sigma factors, we found that, in contrast to bd0881, deletion of which had no effect, the product of gene bd0743 acts more like the heat shock sigma factor RpoE of other bacteria and represses (directly or indirectly) the level of expression of chaperonin genes groES1 groEL (bd0097 and bd0099) in non-heat shock conditions and the level of expression of the groES2 (bd3349) gene under both heat-shock and non-heat-shock conditions. These data and the finding that the groES2 gene is normally expressed in wild type Bdellovibrio only during the late stages of predation (2–4 hours) when the Bdellovibrio are septating and preparing to lyse the exhausted prey bdelloplast, may suggest that a modified chaperonin complex involving GroES2 is used in Bdellovibrio protein expression and folding that occurs at this point. Ascertaining why this is the case requires more chaperone-specific experimentation, beyond the scope of this study and mutagenesis of bd3349 is underway. That the majority of GroES residues shown to interact with GroEL in E. coli are conserved or have conserved substitutions in both of the GroES1 and GroES2 homologues of B. bacteriovorus HD100 supports the idea that they form genuine alternative chaperonin complexes, making GroEL protein folding chambers with different GroES “lids”. It is a tantalising possibility that Bdellovibrio has a requirement for a modified chaperonin complex for the folding of unusual Bdellovibrio proteins required for late-stage prey lysis or Bdellovibrio attack phase cell maturation. The Bd0743-controlled, late-stage expression of groES2 is a possible mechanism for this. Although the (reannotated) Bdellovibrio groES2 gene product is larger at 117 amino-acids than the bd0097 groES1 gene product which is 100 amino-acids, there is no significant additional homology (above that for GroES1) between Bdellovibrio GroES2 and the bacteriophage T4 Gp31 GroES-like protein (data not shown). The bacteriophage T4 Gp31 GroES-like protein allows formation of a larger protein folding chamber for unusual phage capsid protein Gp23 to fold. Bdellovibrio, being a bacterium rather than a phage, does not have any homologues of this protein, so any analogous alternative role for GroES2 in Bdellovibrio protein folding awaits the outcomes of further mutagenesis studies.
Strains and growth conditions
List of strains used in this study
E. coli S17-1
thi,pro,hsdR-,hsdM+,recA; integrated plasmid RP4-Tc::Mu-Kn::Tn7
E. coli DH5α
F’ endA1 hsdR17 (rk-mk-) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacIZYA-argF) U169 deoR (ϕ80dlacΔ(lacZ)M15)
E. coli S17-1: pZMR100
Plasmid vector used to confer Kmr on S17-1 & DFB225 that are being used as prey for KmrBdellovibrio strains
Bdellovibrio bacteriovorus HD100
Bdellovibrio bacteriovorus fliC1 merodiploid
Kmr derivative of HD100 merodiploid for fliC1
Bdellovibrio bacteriovorus bd0743
Bdellovibrio bacteriovorus bd0881
RNA isolation and RT-PCR
List of primers used in this study
Gene knock-out and luminescent prey assay
Kanamycin resistance cassettes were inserted into the rpoE-like sigma factor genes of Bdellovibrio, as described elsewhere [9, 11]. Primers used are listed in Table 3. Luminescent prey assays (with E. coli S17-1 containing the plasmid pCL100) were carried out as described elsewhere [9, 11] except using a Fluostar Optima machine and the final enumeration data were expressed as Bdellovibrio per E. coli. An extra sum of squares F test carried out using the GraphPad Prism 5 software was carried out to show significance.
Electron microscopy and flagella filament length analysis
Bdellovibrio cells were incubated for 24 hours in a predatory culture before being placed on a carbon formvar grid (Agar Scientific), and stained with 0.5% uranyl acetate pH 4.0 as described previously . Cells were imaged using a JEOL JEM1010 transmission electron microscope. Flagellar lengths were measured to the nearest 0.01 μm for an average of 50 cells per strain, error bars show the 95% CI around the mean for each sample as described previously . Student’s t-test was carried out to determine significance of results.
Hobson BacTracker analysis of bdellovibrio swimming speeds
The swimming speed of each Bdellovibrio strain was analysed using Hobson BacTracker (Hobson Tracking Systems, Sheffield, United Kingdom) exactly as described in , including the use of the lower run speed limit of 15 μm/s to reduce the influence of Brownian motion, and accidental tethered-cell-body rotation, on the speed outputs. Cells were pre-grown for 24 hours in a typical 10 ml predatory culture with E. coli S17-1 as prey under the same conditions as for the electron microscopic analysis above. Student’s t-test was carried out to determine significance of results.
RES designed the experiments and co-authored the manuscript. CL performed the RT-PCR and luminescence assays and co-authored the manuscript, RT constructed the mutants and performed RT-PCR, LH performed the electron microscopy and speed measurements. All authors read and approved the final manuscript
- E. coli:
Reverse Transcriptase Polymerase Chain Reaction
Basic Local Alignment Search Tool.
The authors thank Marilyn Whitworth for technical assistance and thank Dr Peter Lund at Birmingham University for helpful suggestions for future GroES2 work. This research was supported by Wellcome Trust grant AL077459 and by Human Frontier Science Programme Grant RGP52/2005.
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