School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK1
Author for correspondence: Christopher M. Thomas. Tel: +44 121 414 5903. Fax: +44 121 414 5925. e-mail: c.m.thomas{at}bham.ac.uk
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ABSTRACT |
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Keywords: bacterial chromosome, active partitioning, parA, parB, stationary phase
Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole
a Present address: Institute of Cancer Studies, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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INTRODUCTION |
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Attempts to obtain deletion mutants of the par genes of Caulobacter crescentus were unsuccessful, perhaps due to their involvement in processes other than chromosome partitioning (Mohl & Gober, 1997 ). However, overexpression of ParA resulted in filamentation of cells and mislocalization of ParB, while overexpression of ParB resulted in 5% of cells within the population being anucleate. Deletion of the parA homologue (soj) in Bacillus subtilis produced no detectable chromosomal partitioning phenotype during vegetative growth (Ireton et al., 1994
), whereas deletion of the parB homologue spo0J gave a phenotype characterized by 12% of the bacterial population being anucleate. Anucleate cell production was also observed in soj/spo0J double mutants (Webb et al., 1998
). Both soj and spo0J deletant strains possess more marked phenotypes with regard to chromosome partitioning during sporulation (Ireton et al., 1994
; Sharpe & Errington, 1996
; Quisel & Grossman, 2000
). However, studies with mutants containing Soj or Spo0J fusions with Gfp did indicate protein localization and movement during vegetative growth, consistent with them being part of an active partitioning apparatus (Sharpe & Errington, 1998
; Marston & Errington, 1999
). In Streptomyces coelicolor, inactivation of parA and parB led to no effect during vegetative growth, but did result in production of anucleate spores during differentiation of aerial mycelia (Kim et al., 2000
).
As all of these studies have involved bacterial species that have relatively complicated survival strategies, we have investigated the par genes in Pseudomonas putida because of its simpler life cycle, similar to many other Gram-negative rod-shaped bacteria. As a model system it is useful because its oriC region has an organization similar to that of most other bacteria whose genomes have been sequenced, including striking similarity to B. subtilis (Fig. 1a) (Ogasawara & Yoshikawa, 1992
), but with clear differences from that of the enteric bacteria. We have therefore generated mutants defective in parA and parB and report their properties. The results suggest that, as in other systems studied so far, the products of these genes are most important under specialized circumstances when the bacteria are undergoing a transition from one survival mode to another, during the entry into stationary phase. We propose that the function of the par system is to tether chromosomes at the cell quarters when partitioning is required in the absence of reinitiation of replication.
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METHODS |
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Recombinant DNA technology.
All restriction and modification enzymes were from MBI (Fermentas) with the exception of T4 DNA Ligase (Gibco). All were used according to the manufacturers instructions. Oligonucleotides for DNA sequencing and PCR were from Alta Bioscience (Birmingham University). DNA sequencing was done by using the BigDye terminator sequencing kit (Amersham Life Sciences). Prepared samples were run on gels by Alta Bioscience (Birmingham University). All electrophoresis equipment and reagents were as previously described (Bignell et al., 1999 ).
Construction of suicide plasmids containing deletions of the par genes.
A new P. putida suicide vector was constructed by ligation of the linker 5'-ATCCCCGGGTCGACCATGGCTGCAGAAGCTTTCTAGAGAATTCA-3' and its complementary sequence, with 5' overhangs of 5'-CCGG-3' at each end, into the XmaI site of pEX100T (Schweizer & Hoang, 1995 ) to create the recombinant plasmid pAKE600 carrying sites for BamHI, SmaI, SalI, NcoI, PstI, HindIII, XbaI and EcoRI as a multiple cloning site with blue/white selection (El-Sayed, 2001
). A DNA fragment from pCAW5.3 (Whatling, 1993
) containing the aph gene from Tn5, conferring kanamycin resistance, was ligated into the HindIII/XbaI sites of pAKE600 to create pCOL500. The par region was amplified from P. putida PaW1 chromosomal DNA using the oligonucleotides parA(b) (5'-GGCGGATCCCATATGGCTAAGGTATTCGCAATCG- 3') and parB (5'-GCGTCGACAAGCTTTCAGCGGATGTGAGCGAG-3'), designed using previously determined sequence data (Ogawasara & Yoshikawa, 1992
), as primers in a PCR. The PCR product was cloned into pUC18 using the EcoRI and SalI sites to receive the insert to give pCOL5. The par genes were also transferred from pCOL5 into pCOL500 using the HindIII and BamHI sites to receive the insert to create the control plasmid pCOL502. The EcoRI site was removed from pCOL500 by EcoRI cleavage, treatment with Klenow DNA Pol I fragment plus dNTPs and religation to give pCOL503, and the par genes were inserted using the same sites as for pCOL502 to create pCOL504. A parB frameshift mutation was created by cleavage of pCOL504 with EcoRI followed by treatment with Klenow fragment and religation to produce pCOL505. The par insert was transferred from pCOL5 using EcoRI and SalI to pCOL5.1 a version of pUC18 that had the EcoO1091 site removed by cleavage, treatment with Klenow fragment and religation. An internal deletion mutation was created in parA of the pCOL5.1 insert by cleavage with EcoO1091, treatment with Klenow fragment and religation. The par insert was transferred to pCOL500 using HindIII and BamHI to create pCOL501. pCOL501 (parA), pCOL505 (parB
) and pCOL504 (control) were transformed into E. coli S17-1 by the method of Hanahan et al. (1991)
and used in a mating with P. putida PaW1. The presence of mutant genes in the chromosome was verified by PCR (as described below) on chromosomal DNA produced from possible recombinant cells. The size of the PCR products was used as a test for parA
strains or cleavage of the product with EcoRI as a test for parB
strains. To make a parAB
suicide plasmid, the parA
insert was transferred from pCOL501 to replace the par insert in pCOL504. The resultant plasmid, called pRAL581, was cleaved with EcoRI, treated with Klenow fragment and religated to introduce a frameshift mutation into parB. This double mutant plasmid was called pRAL58.
Construction of suicide plasmids containing amino acid substitutions in parA.
To create the K16A mutation, a 1·07 kbp fragment corresponding to the first 0·5 kb of the parA gene and an equivalent amount of upstream DNA were amplified by PCR using the primers CM3 (5'-GGAATTCCTGCTCGACAGCCTTAGC-3') and CM4 (5'-CGGGATCCCGTTGTTCAGGCTCAGACG-3'). The PCR product was ligated into pGEM-T easy vector (Promega) to create pGC3-4, which was used as a template in a second PCR. The primers used in this PCR were CM1 (5'-CAGGTGGTTGTGGCGCCCACACCA-3'), which contains changes from the native sequence to introduce a K16A substitution, and CM3. The resultant PCR product was used as a primer in a third round of PCR with CM4 as the other primer and pGC3-4 as template. The product of this PCR was ligated into the suicide vector pAKE604 (El Sayed, 2001 ) to give pWZ1. To create the L138V139-R138R139 mutation a 1·08 kbp fragment, corresponding to about 0·5 kb upstream and downstream of codons 138/139 in parA, was amplified by PCR using the primers CM7 (5'-CGGGATCCCGATGAACTGGTAGCATTG-3') and CM8 (5'-GCTCTAGAGCGAGGCTGATACTTGCCGC-3'), and the product was ligated into pGEM-T easy vector to create pGC7-8. This was used as a template in a second PCR with the primers CM8 and CM6 (5'-GCTGAATGCTCGGCGCGCTTCCG-3') which contain changes from the native sequence to introduce the amino acid changes. The resultant PCR product was used as a primer together with CM7 and pGC7-8 as template in a third PCR and the product of this, containing a mutant version of parA, was ligated into pAKE604 to create pWZ2.
Suicide mutagenesis of the P. putida chromosome.
Saturated cultures of E. coli S17-1 carrying the appropriate derivative of pCOL500 (see above) were mixed with a 10-fold excess of P. putida PaW1 and filtered onto a sterile 0·45 µm nylon membrane which was then placed on an L-agar plate and incubated at 30 °C overnight. The bacteria were then resuspended in 0·85% (w/v) saline before being spread on M9-minimal medium, to select against E. coli, and with kanamycin to select integrants in which the suicide plasmid had recombined with the P. putida chromosome. Putative integrant colonies were purified by streaking and then transferred to L-agar plates containing 5% sucrose, as the suicide vector contains a copy of the sacB gene which is lethal in the presence of sucrose. Colonies which grew should arise from bacteria in which the suicide vector was excised from the chromosome and lost, and thus may have exchanged the mutant copies of the par genes for the wild-type genes. The presence of wild-type or mutant copies of the par genes was checked by restriction enzyme digestion of PCR products generated from the par region.
PCR.
A Hybaid Sprint thermocycler was used to run the PCR and the conditions were as follows: 5 min at 95 °C followed by 2 cycles of 96 °C for 15 s, 50 °C for 30 s and 72 °C for 2 min, then 25 cycles of 96 °C for 15 s, 55 °C for 30 s and 72 °C for 2 min with a final extension step of 72 °C for 10 min. Chromosomal DNA was prepared by picking a colony from an agar plate, washing the cells in 100 µl 0·85% saline and pelleting them by centrifugation. The cells were then resuspended in 50 µl dH2O and boiled for 2 min. The lysate was spun in a microfuge for 5 min and the cleared supernatant used as template in the PCR to check for presence of the mutant par genes. The reaction mix contained 1 µl Taq polymerase (Boehringer), 5 µl Taq polymerase buffer (+15 mM MgCl2), 1 µl template DNA, 2·5 pmol each oligonucleotide primer (Alta Bioscience, Birmingham University), 200 µM each dNTP and dH2O to 50 µl.
Fluorescence microscopy.
The cells were fixed as described previously (Bignell et al., 1999 ), harvested by centrifugation, washed three times in PBS (Sambrook et al., 1989
) and finally resuspended in 200 µl PBS. The cells were added to coverslips coated in poly-L-lysine (Sigma) in 5 mm diameter areas, left for 5 min, aspirated and then washed three times in PBS and air-dried. The cells were rehydrated in PBS, left for 5 min then aspirated and washed three times in PBS. The dried coverslips were mounted as described previously (Bignell et al., 1999
). The slides were viewed using an Olympus IX70 inverted reflected light fluorescence microscope fitted with a Sensys CCD camera (Photometrics). Images were captured and manipulated on a Macintosh G3 with the Smartcapture I program (Digital Scientific). For each culture at each time point images containing
2000 cells were assayed to determine the number of anucleate cells in the population. Two hundred cells chosen at random for each culture at each time point were measured using the IPlab software (Vysis) to determine their total fluorescence, length and width.
Determination of DNA content per bacterium.
The technique used was as described by Burton (1956) . Cells from 4·5 ml culture were pelleted by centrifugation and resuspended in 4·5 ml PBS. Then 0·5 ml 0·5 M perchloric acid was added and the mixture was incubated on ice for 3 h. The mixture was then centrifuged, the supernatant discarded, the pellet resuspended in 1 ml 0·5 M perchloric acid and heated to 70 °C for 30 min before being cooled on ice for 15 min. The mixture was again centrifuged and the supernatant decanted into glass tubes and 2 ml diphenylamine reagent was added [1·5 g diphenylamine (Sigma), 1·5 ml conc. sulphuric acid and 1·5 ml aqueous acetaldehyde (16 mg ml-1) in 100 ml glacial acetic acid]. The mixture was incubated at 30 °C in the dark for 20 h, after which the absorbance at 600 nm was determined. The number of cells per millilitre of the cultures used in the DNA assay was determined by examination of diluted culture samples placed in a Thoma chamber and viewed at x400 magnification with phase-contrast using a Leitz Wetzlab microscope. The two datasets, each with a minimum of five replicates, were used to calculate the DNA per cell of M9-minimal-medium- and L-broth-grown PaW1 cultures. Salmon sperm DNA was used to create a standard curve, but the results are presented as relative DNA content because we are not confident of the absolute values generated.
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RESULTS |
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The mutagenesis strategy had to take into account the polycistronic nature of the gid-par operon of P. putida in which parA is upstream of parB. Fortunately, these are the last two cistrons in the operon. For parA we designed both a deletion and two substitution mutations. The deletion removes 96 bp (32 triplets) of the gene, encoding amino acids 180211 of ParA which is 251 aa long (Fig. 1b). The in-frame nature of the mutation means that polar effects on transcription and translation of parB are unlikely. However, to make the study more complete we also included two additional mutations affecting motifs, located in the N terminus, which are conserved in the parA family. These mutations should have no polar effects at all. Motif I, corresponding to the Walker Box A of ATPases, was disrupted by a K16A amino acid change, and Motif IV, which may be involved in interaction with the cell membrane, was disrupted with simultaneous L138R and V139R amino acid changes. parB was disrupted by means of a frameshift mutation which was predicted to generate a mutant protein identical to ParB for the first 147 aa fused to the short sequence LIRTHPAAGG before terminating prematurely. The remaining segment of ParB should contain neither the predicted dimerization nor the DNA recognition domains (Fig. 1b
) and should therefore be non-functional.
Because knocking out one of a pair of genes that work together may have a different effect from knocking out both, we also created a strain containing both the parA deletion and parB frameshift. This was achieved by simultaneously introducing mutant copies of both par genes into P. putida using a suicide vector containing a doubly mutated par region.
Determination of the phenotypes of parA and parB mutants
To assess the effect of these parA and parB mutations, we first compared the wild-type and deletion/frameshift mutant strains after growth to stationary phase in rich medium (L-broth) at 37 °C. Neither the parA nor the parB mutant strains showed any chromosome loss. However, when we repeated the experiment in M9-minimal medium we observed significant numbers of anucleate bacteria for all the mutants (Fig. 2eh
). The averaged levels of anucleate bacteria from five experiments were as follows: parA
, 9·9±0·9%) and parB
, 4·9±0·8%. This compares with 0·9±0·4% observed for the wild-type control, which in fact corresponded to only very rare appearance of anucleate bacteria (Fig. 2a
d
). The parA
phenotype was significantly and reproducibly more severe than the parB
phenotype. The double parAparB mutant gave 11·6±3·4% anucleate cells averaged over two experiments, similar to the single parA mutant. The point mutations in parA, parA K16A and parA LV138/9RR, gave 10·9±1·5 and 10·8±2·1%, respectively, averaged over two experiments.
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To rule out the possibility that the results presented in Fig. 4(bfigr id="f4">e) represent changes in DAPI permeability, we determined bacterial DNA content by a combination of cell counts and total DNA determination by the diphenylamine reaction as described in Methods. The results (Table 2
) are consistent with the differences in DAPI fluorescence intensities observed in P. putida grown in the two media types and support the proposal that when grown in M9-minimal medium the chromosome copy number of P. putida decreases to one per cell during stationary phase. After growth in L-broth to stationary phase the normal complement appears to be somewhere between one and two chromosomes per cell (Table 2
).
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DISCUSSION |
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The Par- phenotype observed in M9-minimal medium was unexpectedly severe, with up to 14% of bacteria being anucleate in some cultures of parA mutants, when compared to phenotypes previously observed in B. subtilis. The differences in the apparent severity of the Par- phenotype arising from the parA and parB mutations may reflect pleiotropic effects of the mutations, if ParA and ParB affect other aspects of the bacteria. In the RK2 plasmid system we have noted that deletion of the parA equivalent has a greater effect on stability than deleting the parB equivalent (Williams et al., 1998 ; Bignell et al., 1999
). We have suggested that the explanation for this is that, in the absence of ParA, ParB can pair the centromere-like sequences, thus actively interfering with the normal partitioning process (Williams et al., 1998
; Bignell et al., 1999
; Jensen & Gerdes, 1997
; Bignell & Thomas, 2001
). However, the double mutant strain shows a rate of anucleate cell production more characteristic of parA mutants. This indicates that the higher rate of chromosome loss in the parA mutant is not simply due to retention of active ParB.
The fact that we do not see anucleate cells in diluted cultures brought back into exponential phase is presumably due to outgrowth by the normal bacteria and possible lysis of the anucleate bacteria. Indeed the proportion of cells that were anucleate in the parB strain decreased during prolonged incubation in stationary phase. Given that the total number of bacteria did not increase during this time suggests the disappearance of these forms. The differences in the apparent severity of the Par- phenotype arising from the parA and parB mutations may reflect differences in the stability of these forms as well as the rate at which such forms appear.
B. subtilis spo0J mutants exhibit a weak Par phenotype during vegetative, exponential growth (Ireton et al., 1994 ). A very weak Par phenotype is also seen in P. putida for both parA and parB mutants (Fig. 3
). However, the par mutations appear to have no effect on exponential growth rate and expression of the Par- phenotype is not as severe as it is in later stages of the growth cycle. Thus the importance of the par genes during the transition from exponential to stationary phase in P. putida, at least in M9-minimal medium, parallels the importance of the equivalent genes during sporulation in B. subtilis (Ireton et al., 1994
; Sharpe & Errington, 1996
) and S. coelicolor (Kim et al., 2000
). In B. subtilis, the results to date have been interpreted to mean that Soj and Spo0J are needed for chromosome tethering during segregation into the prespore (Sharpe & Errington, 1996
). In S. coelicolor they are needed to ensure that chromosomes are regularly spaced relative to the septa laid down during differentiation of aerial hyphae into spores (Kim et al., 2000
).
The question is therefore, what is the critical process that the par genes are essential for? A cartoon of the divisions occurring during progress to stationary phase is shown in Fig. 5. Fitting our observations to this scheme would have the bacteria grown in M9-minimal medium reaching the stage of a single non-replicating chromosome per cell (Fig. 5c
), whereas the L-broth-grown bacteria would have reached the stage of a partially replicated chromosome being present in each cell (Fig. 5b
). Why should this make a difference in the observed Par- phenotype?
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Second, it may be that the key difference between cells in rich and minimal medium is whether or not, at the final division before entering stationary phase, the chromosomes are still replicating, i.e. whether they are attached to the replication apparatus. The basis for this hypothesis is that DNA replication appears to take place at membrane-associated replication factories located at mid-cell or quarter/three-quarter positions in both B. subtilis (Lemon & Grossman, 1998 , 2000
) and E. coli (Hiraga et al., 1998
, 2000
; Brendler et al., 2000
). Recent work (reviewed by Lewis, 2001
) suggests that the process of replication feeds the daughter molecules out to either side of the replication plane, mediated by proteins such as Smc (Britton et al., 1998
) and SeqA (Brendler et al., 2000
). It has been suggested that the cycle of replication from initiation to termination and reinitiation is more akin to sister chromatid separation during replication than mitosis (Sawitzke & Austin, 2001
). The ability of replicating chromosomes to drive the physical process of separation in the absence of a functional partitioning apparatus, perhaps as a simple consequence of their bulk, is also suggested by the observations of Lin & Grossman (1998
). They observed that while soj/spo0J mutations have a major effect on stability of low-copy-number plasmids whose segregational stability in B. subtilis is dependent on the presence in the plasmid of the putative centromere-like sequence from the chromosome, these mutations have little effect on chromosomal partitioning in B. subtilis. Thus reinitiation of replication prior to cell division may then tether the replicating chromosomes symmetrically on either side of the division plane. Only if this tethering is not in place, as in cells where replication has ceased in preparation for stationary phase, might it be essential to have a partitioning apparatus to ensure that the chromosomes remain on the correct sides of the septum. Therefore, we suggest that the Par apparatus is redundant in exponentially growing bacteria with overlapping cell cycles (Bignell, 1999
).
The above hypothesis makes some testable predictions. For example, it predicts that at very slow growth rates, if reinitation of replication does not take place before division, then we should also see a Par phenotype. Also, if we can block DNA replication, by for example interfering with DnaA function, we would expect to see the Par phenotype exhibited during the run out to non-growing bacteria. Therefore, further observation of the growth conditions under which we observe chromosome loss from these mutants as well analysis of the location of the oriC region in wild-type and par mutants of P. putida during the transition to stationary phase will allow us to test these hypotheses. These hypotheses focus attention critically on attachment of the nucleoid to the cell membrane and the relationship between initiation of replication for the next cell cycle and completion of division for the last cell cycle. Both these areas are aspects of microbial cell biology that deserve further attention.
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ACKNOWLEDGEMENTS |
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Received 22 June 2001;
revised 21 September 2001;
accepted 29 October 2001.