Legal bases for patent and utility model nullity cases are defined under Art. 129 and 165 in the Decree Law #551 pertaining to the protection of patent rights in Turkey.
Art. 129 DL #551 includes a passage in identical context with the provision of Art. 101(3)(a) EPC, stipulating that a patent can be invalidated in part.
Referring to Art. 130 DL #551, a court action must be initiated to request invalidity of a Turkish patent. Such an action can be instituted up to within 5 years after termination of the patent right. Art. 131 DL #551 defines ex nunc effect of a revocation decision (as given in Art. 68 EPC).
The Turkish utility model system has a self-declaratory nature in the sense that no search and examination is conducted. The turbulent nature of the system e is acknowledged by lawmakers in Art. 162 DL #551, explicitly specifying that usefulness of the content of a Utility Model document is not under official guarantee. Utility model applications are subject to prior art search as of 10/01/2017 under the new Industrial Property Law 6769 subject to provisional provisions.
The third party observation system for utility models under DL #551 in Turkey is a hybrid application of Art. 99 and Art. 115 EPC in the absence of an examination or opposition authority. It has similarities to third party observations filed under Art. 115 EPC as it is a pre-grant procedure. A third party can base his observations on conventional grounds similar to those under Art. 100 EPC, with the exception of inventive step requirement, which is not a substantive law requirement for obtaining a utility model grant (Art. 154 DL #551).
Typical grounds for nullity cases specified in Art. 129 DL #551 are identical to the ones defined by Art. 100 EPC and additionally the cases where Art. 52(2) EPC, 53(a) or 53(b) are applicable. The legal implications under Art. 100(b) and 100(c) are relevant according to the DL #551. However, certain legal gaps covered by the Case Law within the European patent law framework do not have clear implementations in Turkey. As a quick example among many others to demonstrate the situation, it is not legally clear whether the content of the priority document forms part of the documents on which an applicant can rely when making amendments to his national application claiming priority of an earlier application.
In response to an infringement action, the alleged infringer may initiate a nullity action and the court where the second action was filed may decide to hear the cases together.
Referring to Art. 129 DL #551, it is similar to Art. 100 EPC providing an exhaustive list of legal situations where revocation of a European patent can be requested. These opposition grounds cover patentability under Art. 52-57 EPC, enablement under Art. 83 EPC and subject-matter extending beyond the content of the application as filed (analogous in the cases of a divisional application or an Art. 61 application) under Art. 123(2) EPC. Similarly, Art. 129 DL #551, citing the same grounds as legal bases for nullity, it adds a fourth ground in which case the right to obtain a patent (entitlement) is in dispute. Art. 129 DL #551 is different from Art. 61 EPC in that it stipulates that a court action can be initiated only by the inventor of the patent, entitlement of which is under dispute, while Art. 61 EPC is silent about this.
The proceedings before the EPO may have a positive or negative impact on the court proceedings in Turkey depending on the arguments of the parties. The legal outcome of the EPO proceedings is legally binding only if a national validation of the same European patent in Turkey is concerned. Otherwise, general patentability criteria and relevance of prior art documents considered before the EPO can be subject to dispute in national court proceedings.
It can be noted that judges usually consider the estimated time amount of the EPO procedure before it is finalized. It is likely that the EPO opposition procedure causes stay of national court procedure. As European appeal procedure takes substantially longer period, the court is more likely to decide itself on the merits of the case.
In terms of the relevancy of certain documents, a court panel is appointed, whose interpretation of the case will be influential. The judges are only legally qualified. The final judgment of a court is mostly in line with the report of the panel of experts in revocation and infringement proceedings. The panels are generally composed of technically and legally qualified members. New panels can be established until at least one additional report supports a given party’s legal position.
As post-grant amendments are not a legal instrument to overcome substantive law objections raised during nullity actions because the patent owner is not allowed to defend his property right by way of limiting its invention. The remaining choice is renouncing one or more claims so that the document would remain valid for the rest of the claims (DL #551, Art. 135). The claim sets of Euroepean patent can be conventionally amended and limited under the EPC. Therefore, the European opposition, appeal and limitation proceedings have a prominent role over the course of court proceedings in Turkey.
However, it is possible to file a notice of opposition in post-grant proceedings as of 10/01/2017 under the new Industrial Property Law 6769, subject to provisional provisions thereof.
DL #551, Art. 129 prescribes that the court can order partial revocation of a granted document in respect of certain claims.


If you or a loved one landed at Los Angeles International Airport, only to be taken into custody while passing through customs because apparently, another state in the United States has a warrant seeking you or a loved one, it can be a confusing and frightening experience.
What procedural requirements must the other state show California before California can let the other state come to “get you?” In other words, what documents must the other state, known as the demanding state, show a California judge ruling on your rights, before the demanding state is allowed to take you into custody there?
The answers are set forth under California law. Before an “asylum state,” i.e. California, can issue an extradition warrant, the judge must have three things to ensure the order to move someone in custody to the demanding state is proper.
About This Article Briefly: There are certain documents that are required for a “demanding state” to bring back a fugitive from an “asylum state.” This article explains what a requisition, affidavit made before a magistrate and a warrant are.
First, the asylum state must receive a formal, written demand for extradition (called a requisition) from the governor (executive authority) of the demanding state (Penal Code § 1548.2 requires this). This demand is often a simple, short demand that is one page in length and given to the prosecutor in the demanding state, who then provides it to the prosecutor in the asylum state.
However, the requisition must be signed by the governor of the demanding state or a person performing the function of the governor (Penal Code § 1548(b)) and should state the name of the person sought and the crime charged or the reason that the person is to be returned to the demanding state.
The requisition must also allege that the case falls within one of the statutory bases for extradition (Penal Code § 1548.2) and that the person sought was present in the demanding state at the time the crime was committed and thereafter fled from the demanding state. If the requisition does not have these two allegations, it is defective and extradition may be successfully resisted (In re Brewer (1943) 61 Cal.App.2d 388, 143 P.2d 33).
If applicable, the requisition should add that the person sought has escaped from confinement or has violated the terms of his or her bail, probation or parole (Penal Code § 1548.2). However, if this allegation is not included, but applies, the requisition is not defective as long as the prior required allegations are included. In re Fedder (1956) 143 Cal.App.2d 103, 299 P.2d 881; Gottfried v. Cronin (Colo. 1976) 555 P.2d 969.
Second, the asylum state must receive a certified copy of an indictment, information, or affidavit made before a magistrate or a certified copy of a judgment or sentence if the person has been convicted (Penal Code § 1548.2).
A complaint, sworn before and signed by a magistrate, is an “affidavit made before a magistrate” (see People v. Mack (1977) 66 Cal.App.3d 839, 136 Cal.Rptr. 283). The definition of a magistrate varies by state. Usually, a magistrate is a judicial officer (see Penal Code § 808), however, in some circumstances, it can be a court clerk or a notary public (see Compton v. Alabama (1909) 214 U.S. 1, 53 L.Ed. 885, 29 S. Ct. 605).
If an affidavit is being used, it should not be made “on information and belief” In re Spears (1891) 88 C. 640, 26 P. 608. It will not be considered defective, however, if the sources of the information and belief are explained or included through supplementary documents such as affidavits or police reports. See In re Cooper (1960) 53 Cal.2d 772, 3 Cal.Rptr. 140.
Third, the asylum state must have a copy of any warrant issued if one was issued, however, if one was never issued, then it is not required (Penal Code § 1548.2; In re Cooper (1960) 53 Cal.2d 772, 3 Cal.Rptr. 140).


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Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.
Replication and segregation are the two major processes needed to maintain a chromosome stably. In bacteria, there has been considerable progress in our understanding of the process of chromosome replication. How the replicated sister chromosomes segregate to opposite cell halves is also becoming clear (62, 73). Other than those of Escherichia coli and its close homologs in the gammaproteobacteria, such as Haemophilus influenzae, most sequenced bacterial genomes have homologs of plasmid partition genes, parA and parB (29). Plasmid partitioning also requires a cis-acting sequence, parS (a centromere analog), to which ParB binds. The parS sequence is also well conserved in bacterial chromosomes (49). The par genes have been studied mostly in low-copy-number plasmids, such as P1, F, and R1, where they confer segregational stability (3, 28, 57). ParA is an ATPase and can be of three types (27). The actin-like ATPase belonging to plasmid R1 polymerizes to form filaments that link at each end with ParB-bound plasmid parS sites. Continued polymerization drives the plasmids to opposite cell poles (13). The second type of ATPase, characterized by Walker-box motifs, is more common. Members of this class of ParA also polymerize but appear to function by different mechanisms. The mechanisms involve a dynamic behavior of the ATPase, which seems to depend on the ParB/parS complex, and in some cases chromosomal DNA that somehow distributes the plasmid copies maximally away from each other, thereby increasing the probability of their presence in opposite cell halves (1, 6, 14, 22, 33, 34, 47, 65, 74). Recently, a third type of par system, which uses tubulin-like GTPases (dubbed TubZ) in plasmid segregation, has been discovered (2, 5, 40, 55).
The chromosomally encoded Par proteins appear to be both homologs and analogs of their plasmid counterparts (29, 30, 48). The functional relatedness of the two became evident when the chromosomal par system of Bacillus subtilis was shown to stabilize an unstable Bacillus plasmid (48). More strikingly, the Bacillus system stabilized an unstable E. coli plasmid in E. coli (77). The latter result implied that no host-specific factors are required for plasmid partition, which is still believed to be the case (26).
Chromosomal Par proteins have been studied in many bacteria, including B. subtilis (35, 48), Burkholderia cenocepacia (19), Caulobacter crescentus (12, 21, 53), Helicobacter pylori (42), Mycobacterium smegmatis (36, 37), Pseudomonas aeruginosa (8, 41), Pseudomonas putida (30, 46), Streptomyces coelicolor (37), and Vibrio cholerae (25, 64). These studies revealed several features of the par genes. (1) They map close to the origin of replication but are not essential except in C. crescentus. (2) Their absence or overproduction causes at least some segregation defect, indicated by increased formation of anucleate cells, abnormal nucleoid compactness, and reduced separation of origin-proximal markers. (3) They contribute to other cellular processes, which include chromosome replication, cell division, and sporulation in B. subtilis (35, 54), cell division in C. crescentus and S. coelicolor (12, 21, 37, 52), and cell motility in P. aeruginosa (41).
Studies in C. crescentus and B. subtilis have contributed the most to our current understanding of chromosomal par systems. In C. crescentus, replication initiates at the stalked pole, and one of the daughter origins moves rapidly to the opposite pole. All three components of the parABS system contribute to chromosome segregation (60, 61, 66). In B. subtilis, the contribution of par to chromosome segregation is not crucial. Deletion of soj (parA) causes no significant partition defect, and deletion of spo0J (parB) reduces the separation of sister origins from 48 to 40% of cell length only (from cell quarter positions toward the cell center) and causes about 0.3% of cells to be anucleate (35, 43, 44). The nucleoid, however, can be abnormal in 20% of cells grown in rich medium (4). Also, when the B. subtilis parS sites are moved away from their natural origin-proximal positions, they no longer localize to cell quarter positions, implying that the par system might not be sufficient for their localization to cell quarter positions (43). In P. aeruginosa and in P. putida, anucleate cells account for up to 10% of the population in the absence of Par proteins (8, 30, 41, 46). The role of par can be more important under certain growth conditions. In P. putida, anucleate cells were seen at higher frequencies when the chromosome copy number was reduced, such as in minimal media or when cells approach stationary phase (30, 46).
Among the roles of par in cellular processes other than chromosome segregation, there is a considerable understanding of how soj and spoOJ contribute to sporulation and how overproduction of soj leads to overinitiation of replication, a process that is normally regulated by spoOJ (44, 54, 58). The replication stimulation is mediated by Soj complexing with the replication initiator protein DnaA (54). SpoOJ, in addition to controlling replication via Soj, helps to promote chromosome segregation by recruiting the SMC condensin near the origin (31).
Both the chromosomes (chromosome I [chrI] and chrII) of V. cholerae have parAB genes and the cognate parS sites (76), which contribute to segregation in a chromosome-specific manner. The par genes of chrI direct the origin of chrI (oriI) to a cell pole, similar to the situation in C. crescentus, and the par genes of chrII direct the origin of chrII (oriII) to the cell center, similar to the situation in E. coli (23, 24, 64, 69). Separate deletions of parAI and parSI of chrI cause oriI to be less polar (25, 43, 64, 76). A previous attempt to delete parBI or parABI has not been successful (64).
Here we have shown that parBI can be deleted and replaced with a drug cassette. The deletion caused little growth defect and some chrI segregation defect, comparable to the defect observed in parAI and parSI deletion strains. Deletion of parBI, however, showed an additional phenotype: parAI-dependent overinitiation of chrI replication. The results are strikingly similar to those found in B. subtilis and provide another example of chromosomal par genes controlling DNA replication.
Strains, plasmids, and growth conditions.Strains and plasmids used in this study are listed in Table 1. E. coli DH5Δlac (BR2846) was used for standard plasmid manipulations and for plasmid propagation. V. cholerae strains were all derivatives of N16961 (CVC209). Bacterial cells were grown in L broth. Antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 25 μg/ml; kanamycin, 25 μg/ml; spectinomycin, 40 μg/ml; and zeocin, 25 μg/ml.
Deletion of parBI.The deletion was achieved by allele exchange, where the gene was replaced with a zeocin (Zeo) drug cassette from the plasmid pEM7-Zeo (68). Briefly, the PCR primers RG135 and RG136, containing XhoI and XbaI-plus-NheI sites, respectively, were used to amplify the C-terminal region of the parAI open reading frame (ORF) (sequence coordinates 2957716 to 2958175). The PCR product was ligated to NheI- and XhoI-digested pEM7-Zeo to obtain pRKG240. A sequence downstream of parBI (coordinates 2956199 to 2956820) was amplified with the primers RG133 and RG142, containing SalI and EcoRI sites, respectively. The product was digested with SalI and EcoRI and ligated to pRKG240, predigested with the same enzymes. The resultant plasmid, pRKG241, was digested with XbaI and SphI, and the fragment carrying the Zeo cassette with parBI flanking sequences was cloned into pDS132 between the XbaI-SphI sites. The resultant plasmid, pRKG242, was transferred by conjugation to V. cholerae CVC209, using E. coli S17λpir (CVC764) as the donor strain. Recombinants in which parBI was replaced with the Zeo cassette without the pDS132 backbone were obtained by serial subculture of the transconjugant followed by sucrose selection. The deletion was confirmed by PCR using the primers RG122 and RG113, internal to the parBI gene, by DNA sequencing, and by Western blotting using antibody against ParBI (see Fig. S1 in the supplemental material). One such construct, CVC784, was used for all subsequent experiments.
Expression of parAI, parBI, and parABI.To express parAI, the gene was amplified by PCR using the primers RG115 and RG214 with Acc65I and XbaI sites at their 5′ ends, respectively, using N16961 DNA as a template. The fragment was digested with Acc65I and XbaI and cloned into similarly digested pBAD24 to make pRN004. An identical procedure was used to express parBI, except the primers were RG5 (with an Acc65I site) and RG6 (with a HindIII site) and the vector was predigested with Acc65I and HindIII. This resulted in pRKG212. The procedure to express parABI was essentially identical to that used for parAI except that the primers were RG115 and RG113, respectively. This resulted in pRN005. ParAI was also produced from pRN006, which was derived from pRN005 by deleting the parBI gene between the sites BxtXI and HindIII. The former site was present in the beginning of parBI and the latter in the vector backbone. ParAI mutants, K16A and D40A, were produced from pJHB41 and pJHB42, respectively, which were derived from pRN006 by site-directed mutagenesis.
Chromosomal integration of P1parS.P1parS as a parS-Kn cassette was inserted at approximately +135 kb in the intergenic region between VC0142 and VC0143. First, the PCR primer pair PS11 and PS14, both containing a PstI site at their 5′ ends, were used to amplify sequences between 133582 and 135584, containing VC0142 and VC0143. The product was cloned into pDS132, predigested with PstI, to generate pPS4. To clone the P1parS-Kn cassette, pPS4 was digested with XmnI and ligated to the parS-Kn cassette obtained from pPS45 by digestion with BamHI and blunting with the Klenow enzyme followed by digestion with SspI. The resultant plasmid, pPS47, was transferred to V. cholerae cells by conjugation, and the transconjugants (CVC851 for wild-type [WT], CVC899 for ΔparAI, CVC853 for the ΔparBI, and CVC1126 for ΔparABI cells) with parS-Kn inserted in chrI were used to localize oriI. To localize oriII, the P1parS-Kn cassette was integrated in the intergenic region between VCA032 and VCA033, both coding hypothetical proteins, as described previously (70). The resultant strains were CVC827 for the WT, CVC1130 for the ΔparAI strain, CVC787 for the ΔparBI strain, and CVC1128 for the ΔparABI strain.
Natural transformation of Vibrio cholerae.A hapR+ derivative of N16961 (CVC1118) was grown to log phase (optical density at 600 nm [OD600] between 0.1 and 0.2) in L broth, and the cells were resuspended in defined artificial seawater (DASW) medium (11). The cells were added to chitin beads (New England BioLabs). After culturing overnight at 30°C to allow biofilm formation, the planktonic bacteria together with medium were removed and replaced with fresh DASW medium. Immediately after the medium exchange, 2 μg PCR DNA fragment from the donor strain was added, and the incubation continued at 30°C overnight. The cells were detached from the beads by vortexing and plated to select for antibiotic-resistant transformants.
Fluorescence microscopy.Cells were cultured in L broth at 37°C to log phase (OD600 between 0.1 and 0.2) and concentrated 100-fold by centrifugation and resuspension in the same medium. They were stained for DNA with Hoechst-33342 at a final concentration of 0.5 μg/ml for 30 s at room temperature and observed under a fluorescence microscope (E1000; Nikon) fitted with a Hamamatsu EM-CCD digital camera (Photonics KK) and controlled by the MetaMorph Premier software program (Molecular Devices, Sunnyvale, CA). Focus locations were measured using the Image J software program (http://rsb.info.nih.gov/ij/index.html).
Flow cytometry.Chloramphenicol (25 μg/ml) and cephalexin (10 μg/ml) were added to exponentially growing cells (OD600 ≈ 0.1) to inhibit new rounds of replication and cell division, respectively. Incubation was continued for an additional 5 h to ensure completion of all rounds initiated prior to addition of the drug. Cells were further treated for flow cytometry, and the number of replication origins in individual cells was determined with a flow cytometer, as described previously (71).
Bacterial two-hybrid assay.A bacterial adenylate cyclase two-hybrid (BACTH) system (EUK001; Euromedex, France) was used according to the manufacturer's protocol. The parAI, parBI, and dnaA genes of V. cholerae N16961 were cloned into pKT25 and pUT18C in frame with the T25 and T18 fragments to make bait and prey plasmids, respectively (Table 1). The PCR primers BJH1 (containing a PstI site at the 5′ end) and BJH2 (containing a BamHI site at the 5′ end), BJH3 (containing a PstI site at the 5′ end) and BJH4 (containing a BamHI site at the 5′ end), and BJH12 (containing a SalI site at the 5′ end) and BJH13 (containing a BamHI site at the 5′ end) (see Table S1 in the supplemental material) were used to amplify the parAI, parBI, and dnaA genes, respectively. The PCR products were digested with PstI and BamHI for parA1 and parB1 or with SalI and BamHI for dnaA and cloned into pKT25 and pUT18C. The pKT25 derivatives carrying parA1, parB1, and dnaA were pBJH3, pBJH4, and pBJH10, respectively, and the corresponding pUT18C derivatives were pBJH5, pBJH6, and pBJH, respectively. Pairs of bait and prey plasmids were used to transform E. coli BTH101 (CVC1837) cells, and the transformants were grown overnight at 30°C in L broth with appropriate antibiotics in the presence of 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). β-Galactosidase activity was measured to determine the functional complementation between the fusion proteins (51).
parBI is dispensable in V. cholerae.Although parAI and parSI have been deleted without any significant growth defect, deletion of parBI has not been successful (64). In the present study, the gene was replaced with a Zeo cassette by an allele exchange method (Materials and Methods). Sequences between coordinates 2956820 and 2957716 covering the entire parBI ORF (2956823 to 2957704) were deleted. The deletion was obtained with similar frequency whether or not a parBI-complementing plasmid (pRKG212) was present in the recipient cells during conjugation and subsequently in transconjugant cells. The parBI gene was also deleted by natural transformation by introducing a PCR fragment (∼2.5 kb) carrying the Zeo cassette and the parBI flanking regions into CVC1118 (N16961 hapR+) and selecting for zeocin resistance. The hapR+ allele was required to make the strain competent for natural transformation (50). The template DNA for PCR was either the plasmid pRKG241 or the genomic DNA of the parBI-deleted strain, CVC784. Similarly, both the parAI and parBI genes were deleted (coordinates 2956820 to 2958505) and replaced with the Zeo cassette by natural transformation using pRKG252 as the template for generating the PCR fragment (∼2.5 kb). The resulting strain was called CVC1124. These results show that ParBI is not essential for V. cholerae growth in L broth.
In an earlier study, codons 11 to 244 of the 258-codon parAI ORF were deleted in frame, and the growth of the mutant was found to be indistinguishable from that of the WT in L broth (64). The doubling time, morphology, and frequency of anucleate cell formation of ΔparBI and ΔparABI cells in L broth were essentially identical to those of the WT and ΔparAI cells, described above. The generation times were approximately 19.5 min, and anucleate cells, analyzed after Hoechst staining, accounted for <1% of the population (varying from 0.1 to 0.8% in the four cases). Nucleoid condensation was also observed at similar low frequencies (0.6 to 0.8%) in all four strains. The parABI genes thus do not contribute significantly to chromosome stability.
oriI segregation is compromised in ΔparBI cells.oriI of V. cholerae preferentially locates to cell poles, but in ΔparAI and ΔparSI cells, the origin is found to be less polar (25, 64). ParBI binds to parSI sites, which are located about +65 kb from the origin of chrI, and ParAI is believed to interact with the ParBI-parSI complex, directing parSI and the nearby DNA, including oriI, to the pole. In this scenario, oriI is expected to be less polar in ΔparBI cells. To test this, WT, ΔparAI, ΔparBI, and ΔparABI cells were marked identically with the plasmid P1parS near oriI, at about the +135 kb position of chrI. These cells were then transformed with pALA2705 to supply GFP-P1ParB under the control of the inducer, IPTG. In this system, GFP-P1ParB binds to P1parS and spreads to nearby DNA to give a bright fluorescent focus. The system has proven to be a reliable indicator of origin number and position in earlier studies (56, 69).
V. cholerae cells grown in L broth at 37°C showed two oriI foci in newborn cells and up to four foci in longer cells (Fig. 1A and B). In WT cells, the focus closest to a pole was on average about 10.8% of the cell length away from the pole; the other origin was at various intermediate positions all the way to the opposite pole (Fig. 1B and 2A). These results are consistent with earlier results obtained with similar and other origin labeling systems (23, 24, 64, 69). In ΔparAI cells, the origin foci were less polar, the mean position of the focus closest to the pole being 17.3% removed from the pole (Fig. 1B and 2A). These results are consistent with earlier findings that suggested a ParAI requirement at the final stages of oriI localization to the pole (25, 64). In parBI and ΔparABI cells, the oriI foci were located 18.9% and 17.3% away from the pole, respectively, similar to the situation in ΔparAI cells (Fig. 1B and 2A). Nucleoid sizes (along the long axes of the cells) were nearly the same (between 98.8 and 99.4% of cell length) in the WT and the mutants. These results support the notion that ParAI and ParBI participate in the pathway that leads oriI to the pole, and nonpolar localization of oriI in the mutants is unlikely to be due to gross changes in nucleoid structure in the mutants.
Location of oriI in WT cells or in parAI-, parBI-, and parABI-deleted cells growing exponentially in L broth. (A) Localization was done by using the GFP-P1ParB/P1parS system by inserting P1parS about 135 kb away (clockwise) from the origin. Cells normally showed up to four foci except in the case of ΔparBI cells, where the number was higher (arrow). These cells accounted for about 7% of the population and were not included in the plot. (B) Plots show the positions of oriI foci in different cells. The cells were arranged according to increasing lengths along the abscissa and focus positions along the ordinate as fractional cell lengths. Focus positions were measured from the pole from which the nearest focus was closer than the nearest focus of the opposite pole. The pole from which measurements were made was placed on the abscissa. Small cells usually showed two foci (light-blue symbols) and larger cells from three to five foci (purple, red, and green symbols, respectively). Black dashed lines show the mean position of the foci nearest to the abscissa from all cells, and it is at 0.108 for the WT, 0.175 for ΔparAI cells, 0.189 for ΔparBI cells, and 0.173 for ΔparABI cells.
(A) Distribution of polar distances (x) of the focus nearest to a pole in the WT and in par mutants. The nearest focus to a pole is shown schematically (black circles) in a two-focus cell and in a four-focus cell. The distribution of the polar distances from different cells is shown after binning of the distances into equal 0.05 fractional cell length intervals and using the mean lengths of the binning distances as the x values (0.025, 0.075, and so on). For ease of presentation, the data were fit by the Kaleidagraph 4.03 software program (Synergy Software, Reading, PA) using unweighted least squares and the fitting function y(x) = c1 xm [exp(−c2x[r]) + c3 exp(−c4 x)], where x denotes the focus position as defined above, y denotes the % of cells, and Ci and m are fitting parameters. r values were 0.998, 0.973, 0.976, and 0.951 for the WT, ΔparAI, ΔparBI, and ΔparABI cases, respectively, and the corresponding locations of the peak maximums were 0.052, 0.118, 0.126, and 0.124. (B) Distribution of cell numbers with different numbers of origin foci. To simplify the distribution for oriI, cells with either one or two foci or three or four foci were grouped before plotting. For oriII, cells had either one or two foci, precluding any need to simplify the plotting further.
Because the segregation defect was modest in the absence of parABI in L broth, we wanted to test whether the defect gets more pronounced during slower growth, as has been reported in P. putida (30). To slow down the growth rate, we used a less rich medium (M63 salts plus 0.2% fructose plus 0.1% Casamino Acids), which increased the generation time of the WT from about 19.5 to 28.5 min. The increases were similar for the three mutants. In all mutants, the oriI foci were removed from the pole compared to their positions in L broth (Fig. 3). In L broth, compared to its position in the WT, oriI in the mutants was on average about 7% more removed from the pole. In the new medium, the corresponding value was 18%. The basis for this growth rate-dependent requirement of par function remains to be understood.
Location of oriI in WT and in parAI-, parBI-, and parABI-deleted cells grown in a defined medium (M63 + 0.2% fructose + 0.1% Casamino Acids). (A) In this medium, cells usually showed from one to two foci (dark- and light-blue symbols, respectively) except in the case of parBI-deleted cells, which showed 12 three-focus cells and 1 four-focus cell (red symbols: about 11% of total cells). Black dashed lines show the mean position of the foci nearest to the abscissa from all cells, and it is at 0.059 for WT, 0.252 for ΔparAI, 0.210 for ΔparBI, and 0.267 for ΔparABI cells. Other details are the same as in Fig. 1B. (B) Distribution of cell numbers with different numbers of origin foci.
oriII locates differently from oriI.In L broth at 37°C, usually one focus is seen near the cell center in smaller cells and up to two foci approaching cell quarter positions in larger cells. These replication and segregation patterns appeared unchanged in the mutants (see Fig. S2 in the supplemental material). We note that because oriII foci do not normally separate as much as the oriI foci, a small perturbation of the segregation pattern in the mutants, to the extent seen for oriI, might not have been apparent in the background of inherent noise in oriII positions. Others have tested oriII positioning and found it to remain unaltered in ΔparAI cells (25, 64).
Chromosome I overinitiates in ΔparBI cells.In the above experiments, although the mean location of origins did not differ significantly between ΔparAI and ΔparBI cells, the distribution of cells with different numbers of oriI foci did vary in the two mutants. In the ΔparBI mutant, there was an increase in the number of cells with three or four oriI foci and a corresponding decrease in the number of cells with one or two oriI foci (Fig. 2B). If it is assumed that origin separation follows soon after replication, this indicates that replication of chrI is initiated earlier in the cell cycle in the ΔparBI mutant than in the ΔparAI mutant. The distributions of the WT and the ΔparABI strain were similar to that of the ΔparAI strain. The increase in the number of cells with more replication origins was not simply due to cell elongation but more likely was due to earlier initiation because the number of origins per cell length increased in ΔparBI cells (Table 2). The distribution of cells with different numbers of oriII foci was similar in all four strains (Fig. 2B).
An independent measure of the origin number was obtained by flow cytometry after replication runout. Replication initiation and cell division were blocked by chloramphenicol and cephalexin, and sufficient time (5 h) was allowed to complete replication elongation (replication runout) in cells that had already initiated replication at the time of drug addition. In the case of the ΔparBI mutant, cells with four genome equivalents of DNA dominated over cells with two genome equivalents, whereas the opposite was true in the other three strains (Fig. 4A). These results suggest that at the time of drug addition, more of the ΔparBI cells had already initiated replication, supporting the microscopy results that suggested earlier replication initiation in ΔparBI cells.
Distribution of DNA contents of cells by flow cytometry after replication runout. (A) DNA contents in WT, ΔparAI, ΔparBI, and ΔparABI cells. (B and C) DNA contents under protein overproduction conditions. In panel B, WT cells either had the vector plasmid pBAD or the parAI, parBI, or parABI genes under the control of the PBAD promoter of the vector. In panel C, the plasmids were the same but the host was ΔparABI cells. One hundred thousand cells were analyzed in all cases.
Chromosome I overinitiation requires ParAI.As shown in Fig. 4A, the overinitiation phenotype of ΔparBI cells was not seen in the ΔparABI double mutant, indicating that ParAI could be required for the phenotype. To test this, we overproduced ParAI, ParBI, and the two together in WT and in ΔparABI cells. Overproduction was achieved by cloning the genes in a pBAD vector, under the control of an arabinose-inducible promoter, PBAD, and an increase in the level of proteins of interest was confirmed by Western blotting (see Fig. S1B and C in the supplemental material). In the WT background, the profiles of fluorescence intensity were comparable between the cells with pBAD and cells with pBADparBI (Fig. 4B). In contrast, when the cells had the pBADparAI or pBADparABI overproducing plasmid, there were more cells showing higher fluorescence, i.e., there was a decrease of cells with two genome equivalents and an increase of cells with four genome equivalents. In the ΔparABI background, the results were essentially the same, with the profiles of cells with pBAD and pBADparBI being comparable to each other and the profiles of cells with pBADparAI and pBADparABI being comparable to each other. A common feature of these results is that overinitiation could be seen under all ParAI overproduction conditions, whether or not ParBI was also overproduced. The role of ParBI in this process is most likely that of an inhibitor of ParAI activity rather than a transcriptional repressor of the parAI gene, since overinitiation was achieved in ΔparBI cells without significant overproduction of ParAI (see Fig. S1A). The overinitiation activity of ParAI apparently dominated over the inhibitory activity of ParBI when both proteins were overproduced. This could be because in these experiments ParAI was overproduced about 8-fold more than ParBI (∼160- versus ∼20-fold, respectively; see Fig. S1). When these findings are taken together with the microscopy results, it is clear that ParAI is required to increase initiation from chrI. This can happen at near-physiological levels of ParAI, but the level needs to be significantly increased when ParBI is also present. When ParA1 was overproduced about 30-fold (using 0.02% arabinose), the replication enhancement was not apparent (data not shown). We note that even in the absence of ParBI, a considerable excess of ParAI over the physiological level was needed for overinitiation when ParAI was supplied from a plasmid rather from its natural chromosomal locus. The basis of this cis-trans difference in the efficacy of ParAI remains to be understood.
From studies in B. subtilis, it was expected that the nucleotide-bound state of ParAI could be important for its role in replication initiation (20, 54). As a preliminary test of this notion, parAI was mutated at conserved positions that resulted in K16A and D40A changes in the protein. The K16A change is expected to be defective in ATP binding and the D40A change in ATP hydrolysis (54). As in B. subtilis, only the latter protein was proficient in replication promotion (Fig. 5). In summary, both the amount and form of ParAI appear important for the proper control of replication initiation of V. cholerae chrI.
Overreplication phenotypes of ParAI mutants with K16A and D40A changes in Walker-box motifs. (A) Replication was determined by flow cytometry as with Fig. 4. (B) The protein level after overproduction was determined by Western blotting, as with Fig. S1 in the supplemental material, and the levels in the mutants were comparable to that of WT ParAI.
ParAI overproduction causes nucleoid condensation.Cells overproducing ParAI, ParBI, or both were also examined by microscopy. In WT cells without the overproducing plasmids, the nulceoid boundaries spanned 91% ± 4% of the cell length (Fig. 6). The results were essentially similar in the ΔparAI cells, where the value was 94% ± 4%. In contrast, under ParAI-overproducing conditions, the nucleoid appeared more compact, since the value was 62% ± 16%. When ParBI or both ParAI and ParBI were overproduced, the compaction was again not significantly different from that of the WT (96% ± 4%, 90% ± 11%, and 91% ± 4%, respectively). It appears that ParBI can effectively suppress the nucleoid compaction phenotype of ParAI.
ParAI overproduction causes nucleoid condensation and positions oriI at the pole-ward edges of the nucleoid. The nucleoids were visualized by Hoechst staining, and oriI was visualized by marking with GFP, as in Fig. 1. Overlays of the two are shown in the last column. The positions of nucleoid boundaries and oriI were measured independently and are shown together in the same plots to visualize the correspondence between the two positions on a population basis, not on a cell-to-cell basis. The color coding is the same as in Fig. 1, except the nucleoid boundaries are shown in dark blue. The results are summarized in a schematic where the nucleoid is shown as a blue oval and oriI positions as red circles.
We also checked oriI localization in each of the overproducing strains. The position of the origin was less polar in ParAI-overproducing cells, comparable to the situation in ΔparAI cells (Fig. 6). In the former case, it is likely that the origins failed to reach the pole or stay at the pole because of nucleoid compaction. If there exists an anchoring mechanism to hold the origins to the pole, as has been found in C. crescentus and sporulating B. subtilis cells, it might not have been strong enough to withstand the force of compaction or was made ineffective by an excess of ParAI (9, 12, 21). It is intriguing that both the absence of a par system (ΔparABI and ΔparSI) and overproduction of ParAI lead to the same oriI localization defect, although the reasons in the two cases need not be the same.
In B. subtilis, increased expression of Soj makes the nucleoid less compact, the opposite of the results presented here (58). Compaction was seen when both Soj and SpoOJ were overproduced together. Under these conditions, the SpoOJ-to-Soj ratio was higher than their ratio in the WT, which was interpreted to mean that the excess of SpoOJ could be responsible for the compaction. SpoOJ has been found to recruit the SMC protein, a condensin (31, 72). In C. crescentus, constitutive overexpression of either ParA or ParB causes cell division defects that are less severe when both proteins are overexpressed together (53). These results suggest that proper stoichiometry of the two Par proteins is important for normal cell physiology. The importance of concentration is also suggested by the observation that Soj can be either a positive or negative regulator of replication, depending upon its level of production (54).
Interactions of DnaA with ParBI and ParAI.The DnaA protein is considered to be the limiting factor for chromosomal replication initiation (63). Therefore, it is expected that ParAI-mediated stimulation of replication could be mediated through DnaA. Such has been the observation for B. subtilis (54). To determine whether this could be the case in V. cholerae, we carried out protein-protein interaction studies using ParAI, ParBI, and DnaA. The interactions were assayed by the bacterial two-hybrid system. The system is based on the reconstituted activity of Bordetella pertussis adenylate cyclase (cya) from its two complementary fragments, T25 and T18 (38). The full-length ParAI, ParBI, and DnaA proteins from V. cholerae were fused at the C terminus of either the T25 or T18 fragment. As a positive control, a 35-amino-acid (aa)-long leucine zipper belonging to the yeast transcriptional activator GCN4 was fused to both T18 and T25. Considering the activity obtained with two empty vectors (about 20 Miller units) as the background level, 10/10 randomly picked colonies containing the leucine zippers as interacting partners showed at least 2-orders-of-magnitude-higher activity. In the experimental samples, 2 to 5 colonies were considered significantly positive, whereas in strains containing one empty vector plus one with a fusion protein, none of the colonies showed activity significantly above the background level (Table 3). The assay thus indicates interactions between ParAI and ParAI, ParAI and ParBI, and ParBI and ParBI, although the interactions were much weaker than those between the leucine zippers. In the case of DnaA, an indication of interactions with both ParAI and ParBI could be found when DnaA was fused to T18 (T18-DnaA). However, with T25-DnaA, the results were positive with T18-ParAI only. T25-DnaA fusion, however, complemented to some extent the growth defect of a dnaA46(Ts) strain at the nonpermissive temperature (see Fig. S3 in the supplemental material). It is possible that the conformation of DnaA upon fusion to T25 could have changed, so that it was no longer active for interaction with itself and ParBI. Alternatively, the concentration of T25-DnaA could be lower than optimal because of the plasmid producing the fusion protein having a lower copy number than the one producing T18-DnaA.
We also tried to validate these interactions by the yeast two-hybrid system. Here, the evidence for positive interactions was obtained among the three proteins in all possible combinations (see Table S2 in the supplemental material). However, the results were again significantly above the background level in only some of the colonies. At least in the bacterial system used, the all-or-none response is expected if the proteins interact weakly. The proteins are expressed from the lac promoter, which itself depends upon cAMP. If the basal level of expression of the bait and prey proteins is low and the proteins interact weakly, this might not lead to enough cAMP production, and a negative result follows. When the basal level of cAMP crosses a threshold, more bait and prey proteins are made, which in turn make more cAMP, leading to an autocatalytic reaction and a robust positive response. Another inherent weakness of the system is that it is based on multicopy vectors, and plasmid copy number fluctuation could also be a source of heterogeneity. In our case, in the extract from the cya mutant host, the fusion proteins were barely detected by Western blot analysis, although all the fusion constructs were transcribed (see Fig. S4A). The ParAI and ParBI fusion proteins were detected using a cya+ strain, which was also deleted for the ClpXP and Lon proteases (see Fig. S4B). Unfortunately, DnaA fusion proteins could not be detected using antibodies against E. coli DnaA. Thus, both low expression and instability of the fusion proteins could be responsible for the all-or-none response in the results of the bacterial two-hybrid system.
In summary, the results from bacterial and yeast two-hybrid systems indicate that V. cholerae ParAI, ParBI, and DnaA can interact with each other. Interactions between the ParA and ParB proteins have been found in other bacteria (7, 8, 10). DnaA interaction with ParA (Soj) has been found in B. subtilis (54). To the best of our knowledge, it has not been reported that a ParB protein (ParBI) can also interact with DnaA.
Concluding remarks.V. cholerae provides a second example of participation of chromosomal Par proteins in chromosomal replication, which was first observed in B. subtilis (44, 54, 58). Conservation of Par functions in different bacterial phyla was evident from sequence conservation, stabilization of a Bacillus nato plasmid by the B. subtilis par system, and even stabilization of an E. coli plasmid by the B. subtilis par system in E. coli (29, 48, 77). The Par proteins, however, are not required for chromosomal replication initiation in either B. subtilis or V. cholerae and normally do not appear to contribute to replication significantly under the conditions tested. In E. coli, the regulators of oriC include DnaA, SeqA, Dam, pgsA, DnaK, relA, Hda, IHF, HU, FIS, IciA, datA, DiaA, nrdAB, fre, and DARS1 and -2 (39). Most of these regulators can be deleted at least individually with minor effects. In a system with multiple regulators, the effect of any one may not be expected to be great. Even then, it is remarkable that the apparent ancillary role of ParA in stimulatnig replication has been retained in two bacteria that are believed to have diverged more than a billion years ago.
Replication and segregation modules in plasmids are usually found next to each other. However, it is not known even for well-characterized systems whether the two modules can function mechanistically independently of each other in their natural context (27). In only a few cases, the modules are not discrete and some links between the two systems have been found at the level of transcription. For example, in the broad-host-range plasmid RK2, the replication initiator gene trfA is controlled by the ParB homolog KorB, which also serves as a general transcription factor in that plasmid (67). In the repABC plasmid family, the partition and replication genes are present in the same operon (15). In a Streptococcus pyogenes plasmid pSM19035, the replication and partition genes are under a common regulator, ω2 (16). In the linear phage-plasmid N15, one of the two promoters of the partition operon is repressed by protelomerase, the enzyme that completes prophage replication (18). Beyond transcription, however, there is no evidence yet as to whether the processes of replication and partition influence each other. The situation seems to be different for chromosomes. The act of replication has been invoked to provide the driving force for segregation (45). A negative regulator of replication, SeqA, has been proposed to help in proper segregation of daughter chromosomes (75). Here we show the opposite in that ParB contributes to chromosome segregation but can be a negative regulator of replication as well (Fig. 1 and 4). In B. subtilis, SpoOJ helps to regulate chromosome replication via Soj and chromosome segregation by SMC condensin recruitment near the origin of replication (31, 72). V. cholerae encodes a MukBEF complex, analogous to SMC. It remains to be seen whether ParBI helps recruit the complex near oriI. Together these results indicate that interactions between replication and segregation systems might be more common than is currently known. Finally, we note that V. cholerae ParAI could not stimulate replication in E. coli or in an engineered E. coli strain where oriC has been replaced with V. cholerae oriI (R. Kadoya, unpublished results) (17). Thus, even in closely related strains, such as E. coli and V. cholerae, there seems to have been considerable adaptation between the replication and segregation proteins.
We are grateful to Melanie Blokesch, Ranajit Ghosh, Lyn Sue Kahng, Jon Kaguni, Mike Maurizi, Ole Skovgaard, Preeti Srivastavaand, and Matt Waldor for strains, reagents, and advice. Special thanks go to Aurelia Battesti and Tapan Som for help with two-hybrid systems and to Jean-Philippe Castaing, Grazyna Jagura-Burdzy, David Lane, Michael Yarmolinsky, and the anonymous reviewers for critical reading of the manuscript.
This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute.
Accepted manuscript posted online 21 January 2011.
† Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01067-10.
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Calcium chloride minerals in the natural state are rare and only found in a few specific evaporite associations. On the other hand, calcium chloride-rich brines are commonplace in the burial diagenetic realm, especially in deep high-salinity basinal brines and in a number of hypersaline lake waters, especially in rift settings. In the subsurface, these brines also play a significant role in the formation of a number of metal ores. Occurrences of both the brine and the minerals have significance in modelling rock-fluid interactions and seawater chemistry across geological time.
At earth-surface temperatures, calcium chloride can exist in the solid state as the anhydrous form (CaCl2) as well as in four levels of hydration – CaCl2.H2O; CaCl2.2H2O; CaCl2.4H2O; CaCl2.6H2O (Table 1). Of this group, CaCl2 occurs naturally as two rare minerals; antarcticite and sinjarite. All of the early studies on calcium chloride and its hydrates were done with laboratory-prepared samples of brines and hydrates, since CaCl2 was not produced on a commercial scale until after the ammonia–soda process for the manufacture of soda ash (Solvay Process) was in operation. Before its industrial uses were discovered, calcium chloride was considered a waste product of brine production. Today, its primary industrial use is predicated on the very high enthalpy change of solution, indicated by considerable temperature rise accompanying dissolution of the anhydrous salt in water (Table 1 – Heat of solution in water). This property is the basis for its largest scale application, namely road de-icing.
In the natural state, most CaCl2 occurs in solution in basinal waters in sedimentary basins and modified pore waters in specific hydrothermal associations. Calcium chloride, in a mineral state in the natural world, occurs as the rare evaporite minerals; sinjarite (CaCl2.2H2O) and antarcticite(CaCl2.6H2O). The related potassic and magnesian calcium chloride minerals, chlorocalcite (KCaCl3) and tachyhydrite (calcium magnesium chloride, CaMg2Cl6•12H2O) are also rare in the sedimentary realm, and have particular evaporite associations and implications (see Part 2).
Outside of an industrial byproduct of the Solvay Process, most CaCl2 is derived from the processing of hypersaline basinal brines. The only current commercially, exploited natural CaCl2 surficial brine source is in Bristol Dry Lake, California (Figure 1). In the USA, for example, basinal brines are the primary commercial source of calcium chloride. Some of these brines in Michigan, Ohio, West Virginia, Utah, and California contain >4% calcium, with the Michigan Basin as the dominant producer. In the USA, a former commercially important source of calcium chloride was as a by-product of the Solvay Process used to produce soda ash. Because of environmental concerns and high energy costs, the Solvay Process has been discontinued as a source of CaCl2 in the USA.
This article will focus on the utility and geological significance of CaCl2 brines, while the next will focus on the geochemical significance of various calcium chloride minerals in particular evaporitic settings.
Calcium chloride depresses the freezing point of water, and its principal use is to prevent ice formation, especially on winter roads. Calcium chloride released to the environment is relatively harmless to plants and soil in diluted form. As a de-icing agent, it is more efficient at lower temperatures than sodium chloride. Solutions of calcium chloride can prevent freezing at a temperature as low as -52 °C (Figure 2). Hence, more than 50% of world CaCl2 usage is for road de-icing during winter. The second largest application of calcium chloride brine exploits its hygroscopic properties and the tackiness of its hydrates. In summer, it is used for roadbed stabilisation in unsealed roads and as a dust palliative. When sprayed onto the road surface, a concentrated CaCl2 solution maintains a cushioning layer on the surface of dirt roads and so suppresses formation of road dust. Without brine treatment dust particles blow away, eventually larger aggregate in the road also begins to shift around, and the road surface breaks down. Using calcium chloride reduces the need for grading by as much as 50% and the need for fill-in materials as much as 80%.
Calcium chloride’s low-temperature properties also make it ideal for filling agricultural implement tyres as a liquid ballast, aiding traction in cold climates. It is also used as an accelerator in the ready-mix concrete industry, although there is concern about its usage because of possible long-term chloride-induced corrosion of steel in highways and buildings. Calcium chloride is also widely used to increase mud fluid densities in oil- and gas-well drilling. It is also used in salt/chemical-based dehumidifiers in domestic and other environments to absorb dampness/moisture from the air.
Calcium chloride is used in the food industry to increase ﬁrmness of fruits and vegetables, such as tomatoes, cucumbers, and jalapenos, and prevent spoilage during processing. Food-grade calcium chloride is used in cheese-making to aid in rennet coagulation and to replace calcium lost in pasteurisation. It also is used in the brewing industry both to control the mineral salt characteristics of the water and as a basic component of certain beers.
Generally, CaCl2 brines are found in permeable strata either below, adjacent to, or above evaporite deposits, gradually becoming more dilute as brines approach the surface, and modiﬁed somewhat in proportion to distance from a potash or salt layer (Figure 1; Table 2). Other natural calcium chloride brines are derived from hydrothermally-modified marine waters. Dilute calcium chloride brines are also occasionally found in coastal aquifers, and some oil or gas formation waters that have been formed from seawater, possibly by a dolomitization reaction supplemented by the leaching of certain types of rocks (Garrett, 2004).
Basinal brines are chemically similar to CaCl2 brines forming hydrothermally at modern mid-ocean ridges, where seawater is being converted by interaction with basalt at elevated temperatures into low-salinity Na-Ca-Cl brines, depleted in Mg and SO4. These CaCl2 waters occur in and near active fracture zones, wherever seawater interacts with labile basalt (oceanic crust) at elevated temperatures and converts the circulating fluid from a Na-Mg-Cl water into a low-salinity Na-Ca-Cl brine, depleted in Mg and SO4. Similar hydrothermally-driven alteration of continental basalts via deeply circulated seawater interactions forms modern CaCl2-rich brine seeps, for example, within the thermally active continental Danakil rift valley (Hardie, 1990; Warren, 2016).
Calcium chloride is produced in commercial amounts using a variety of procedures: 1) refining of natural brines, typically with heating to increase concentration, 2) reaction of calcium hydroxide with ammonium chloride in Solvay soda ash production, and 3) reaction of hydrochloric acid with calcium carbonate. The first two processes account for over 90% of the world’s total calcium chloride production. Historically, natural brines sources are the dominant CaCl2 source. There is currently an excess of capacity in the calcium chloride industry, a situation which is only expected to become more acute as synthetic and byproduct capacity increases.
This Silurian halite/potash basin has many aquifers with calcium chloride brines, both above and below the Silurian Salina Group’s halite and potash levels (Figures 3, 4; Garrett, 2004). Major aquifers are the overlying Devonian carbonate and sandstone beds, with many lesser aquifers. In the first porous bed above the potash (Sylvania Sandstone Formation) there is an extensive area of rich calcium chloride brine sitting directly above the potash deposit and extending to the south-southeast. Brine concentrations at nearly the same concentration as potash end liquor in fractures in the intrasalt carbonates. The less voluminous sandstone of the Filer Formation to the northwest contains a similar, but slightly more dilute CaCl2 brine. Several thinner and less abundant aquifers also occur under the potash beds with equally strong, or stronger calcium chloride brines (Figure 3).
The porous 0-90 m thick Sylvania Sandstone lies at the base of the Detroit River Group and is the main source of CaCl brine production. It is in direct erosional contact with the salt succession (Figure 4). The remainder of the Group consists of 0-350 m of variably porous carbonates (Garrett, 1995, 2004). Both sandstones and carbonates contain CaCl2-rich brines and extend across some 40% of the Michigan Basin at depths of from 300-1,400m. Brine concentration and the relative amount of CaCl2 increases with depth. Typically, the brines are only considered to be of economic importance below about 880 m depth. In carbonate hosts, the CaCl2 content varies from 3-23% and KCl from 0.2-2.9%, usually increasing in concert with concentration, as the NaCl content decreases. CaCl2 content in the Sylvania Sandstone varies from 14-22%, KCl from 0.6-2.1%, and both are more uniformly concentrated compared to the carbonate-hosted brines (Garrett, 2004).
Each aquifer entrains roughly the same ratio of ions, but pore waters become progressively more dilute as beds approach the surface about the basin margin. It seems likely that in this basin, a potash liquor originally seeped through and under the potash deposit (and reacted with calcite) was much later forced from its original sediments into the overlying porous strata into the overlying porous strata as they were compressed by deep burial, possibly aided by load-induced pumping induced by the waxing and waning of thick glacial ice that formed over this basin (McIntosh et al., 2011). Variable ionic content, as seen in Table 2, results from their considerably different migration history and variable dilution by meteoric or other groundwater (as is strongly indicated by the brine’s deuterium and 18O analyses), precipitation (such as gypsum), and their different contact with rocks that they could partially leach or react with. However, in the Michigan Basin these reactions were limited, since the porous carbonate strata (average, 20%) contains fairly pure carbonates, and the sandstone strata fairly pure silica (quartz arenites) cemented by dolomite or quartz (Martini, 1997).
There is a general synclinal structure to the strata under the Michigan Basin, and examples of the specific stratigraphy to the southeast of the centre of the basin at Midland are shown in Figure 4. The Detroit River Group consists of 0–350 m of variable porosity carbonates, and at its base there is 0–90 m of porous sandstone called the Sylvania Formation. Each of these formations cover about 40% or more of the Michigan Basin, and contain strong calcium chloride brines at depths of 300–1400 m. Their brines have been commercially recovered in the past, and were generally only considered to be economic below about 880 m. The brines’ total dissolved solids (TDS) and the amount of CaCl2 increases fairly consistently with depth from 3 to 23% CaCl2, and the NaCl and MgCl2 concentrations vary inversely with the CaCl2 . In the Sylvania Formation, the CaCl2 usually ranges from 14 to 22% (Figure 3). Additional information on the brine in other aquifers and the various reactions and changes that have occurred with them are discussed in Martini a(1997), Wilson and Hewett, (1992) and Wilson and Long (1992).
The Michigan Basin brines’ very low pH (4.5 to 5.3) helps to explain an ability to leach and react with other rocks, as is indicated by their high contents of strontium, barium and metals, much of the Sr and Ba probably came from the reaction with calcite. Geothermal water also probably mixed with some of the formations, as indicated by the variable presence of iodine, boron, lithium, caesium, rubidium and other rare metals. With most of the brines, the calcium concentration is somewhat higher than its magnesium equivalent in seawater end-liquor from a potash deposit, and the potassium a little lower. Wilson and Long (1992) speculated that this occurred by the conversion of the clays kaolinite and smectite to illite: Small amounts of glauberite (CaSO4.Na2SO4) and polyhalite (2CaSO4.K2SO4.MgSO4; have also been found in the basin. Finally, some of the calcium chloride aquifers have a slightly elevated ratio of 87Sr/86Sr (range from 0.7080 to 0.7105; seawater is 0.70919), further indicating that there was some rock leaching during burial(Martini, 1997).
What does a CaCl2 basinal brine indicate?
Pore fluids in the deeper parts of many sedimentary basins, especially if they contain a significant unit of evaporite, tend to be CaCl2 brines, entraining large volumes of hypersaline brine and in places, hydrocarbons (e.g., Michigan Basin, the U.S. Gulf Coast, European North Sea Basin, Western Canada Basin and Volga Basin).
Worldwide, one of the principal geochemical characteristics of saline waters in sedimentary basins is the progressive shift in their major ion composition from Na–Cl to Na–Ca–Cl to Ca–Na–Cl dominated waters with increasing chlorinity or salinity (Hanor and McIntosh, 2006). Such basinal brines (also called oil-ﬁeld brines or formation waters) with significant calcium chloride contents have salinities that typically range up to 300,000 mg/l (Hanor, 1994; Lowenstein et al., 2003). The majority of these basinal brines are chemically distinct in their high Ca concentrations, separating their hydrogeochemistries from modern seawater and other common surface and near-surface waters which tend to be Na-Cl-SO4, Ca-HCO3, or Na-CO3 types (Drever, 1997).
Calcium levels in a CaCl2 basinal brine typically exceed the combined concentrations of SO4, HCO3, CO3 ions, (specifically, mCa > ∑(mSO4 + 1/2mHCO3 + mCO3); Lowenstein et al., 2004). And yet, the evaporative concentration of modern seawater leads to brines depleted in Ca, as required by the principle of chemical divides (CaCO3 and CaSO4 divides) for any evaporating water (Hardie and Eugster, 1970). Explanations for the origin of CaCl2 basinal brines remain problematic.
There is no simple pathway by which modern seawater, and most other surface and near-surface waters trapped in sedimentary deposits, can be converted to CaCl2 basinal brines during burial, without invoking significant rock-fluid interaction. Historically, before micro-inclusion studies of chevron halite showed that the ionic proportions of seawater likely varied across the Phanerozoic, the various CaCl2 basinal brines occurrences, for example in in Silurian and Cretaceous age strata were explained as an indicator of widespread dolomitisation and other diagenetic reactions, which preferentially extracted magnesium from pore waters (Garrett, 2004).
Since the mid-1990s, others have argued that CaCl2 enrichments in many ancient basinal brines with thick evaporites in the stratigraphy, including brines in the Detroit Group, are partial leftovers from times of CaCl2-enriched seawater chemistries (Table 3; Lowenstein and Timofeeff, 2008; Lowenstein et al., 2014). That is, Ca-enriched (MgSO4-depleted) pore brines adjacent to thick evaporites are indicators of ancient CaCl2 oceans, with the pore brines being remnants from the time the enclosing evaporitic and marine sediments were deposited (relict or connate brines).
Other authors, such as Houston et al. (2011), conclude this is not necessarily so; they agree that there are two end-members typifying highly saline subsurface brines (MgSO4 depleted or enriched). But, they conclude that end member chemistries relate to either substantial subsurface halite dissolution, or to preservation of early reflux-related seawater. Houston et al. (2011) go on to argue that CaCl2-enriched formation water chemistries from many basins worldwide, including the Michigan Basin, do not support an interpretation of variation in ionic proportions in seawater across the Phanerozoic. They find that CaCl2-rich brines formed either by dissolving bittern salts in the subsurface, or simply lost water in the subsurface after significant rock-fluid interaction had taken place. Water loss might be achieved by interaction with a gas phase at the elevated temperatures of deep burial or, alternatively, water may have been lost to clays. Both these mechanisms would have the effect of dehydrating (concentrating) the brine.
Calling upon a CaCl2 seawater source as an explanation for the origin of basinal brines was criticized also by Hanor & McIntosh (2006) who pointed out that no matter what the starting composition of a paleoseawater, signiﬁcant diagenetic alteration must have occurred to produce the present major ion chemistry of Illinois and Michigan basin brines speciﬁcally, and basinal brines in general. In their view, the diagenetic mineral–brine interactions that occur during burial mask any original compositional variations in the starting seawater.
Hanor & McIntosh (2006) also argued that due to ongoing fluid escape and crossflow, it is difﬁcult, if not impossible, to assign speciﬁc ages to basinal brines in a sedimentary basin. If the age of a basinal brine is not known, then the possible parent seawater, whether CaCl2 or MgSO4 type, cannot be determined. Hanor and McIntosh (2007) illustrated further complications in the interpretation of the timing of the origin of basinal brines. They showed that some brines in the Gulf of Mexico basin were not formed during the Middle Jurassic, contemporaneous with deposition of the Louann Salt, but formed during the Cenozoic from the dissolution of the Louann salt.
Interestingly, many marine potash deposit end-liquor brines have a high to medium–high lithium content, such as the Angara-Lena basin, Russia’s 1600–1900 ppm, the Paradox Basin’s 66–173 ppm Li, the Michigan Basin’s Sylvania Formation’s 36–72 ppm and the English Zechstein Formation’s 7– 65 ppm, etc. (Garrett, 2004)). However, some end-liquors have only a nominal lithium contents, such as from the Saskatchewan, Canada potash deposits. A few calcium chloride lakes also have medium–high values, such as the Don Juan Pond’s 235 ppm, Bristol Lake’s 30–108 ppm, Cadiz Lake’s 20–67 ppm, and Lake Vanda’s 27 ppm (Figure 1). We shall come back to this topic in a future article that will focus on lithium-rich brines.
So, currently, there are two schools of thought used to explain the origin of CaCl2 basinal brines in evaporitic basins. One school assumes that the chemistry of the world’s ocean and its ionic proportions have remained near constant across the Phanerozoic. To form a CaCl2 enriched basinal brine then requires substantial subsurface rock-fluid interactions, utilising mechanisms and processes that include nonmarine parent waters, diagenetic alteration, pervasive dolomitization of carbonates, or bacterial sulphate reduction. All these mechanisms can reduce the proportion of Mg, HCO3 and SO4 relative to Ca in subsurface pore waters (Hanor and McIntosh, 2006). Proponents of this school tend to base their argument on basin-scale variations in the hydrogeochemistry of pore fluids.
The other school (mostly based on the micro-inclusion chemistry of chevron halite) argues for long-term changes in the major ion chemistry of seawater (Table 2). For example, Upper Jurassic, Cretaceous, and Cenozoic seawater records a systematic, long-term (>150 My) shift from the Ca2+ - rich, Mg2+ - and SO42- - poor seawater of the Mesozoic (“CaCl2 seas”) to the “MgSO4 seas” (with higher Mg2+ and SO42- > Ca2+) of the Cenozoic (Lowenstein and Timofeeff, 2008; Lowenstein et al., 2003, 2014). Over that period, the Mg/Ca ratio of seawater rose from 1 in the Early Cretaceous, to 2.3 in the Eocene, and 5.2 in present-day seawater.
Suggested drivers of long-term variation in the major ion chemistry of seawater include; ﬂuctuations in the volume of discharge of hydrothermal waters from the global mid-ocean ridge system (Hardie, 1996), changes in the rates of volcanic activity and weathering processes, and variations in the amount of dolomite formed in the oceans (Holland and Zimmermann, 2000).
In my mind, much of the conflict between to two schools of thought as to the origin of CaCl2 basinal brines stems from the source of evidence. One approach utilises micro-inclusion chemistry in halite chevrons to define the evolution of Phanerozoic seawater. This data is extracted from an intra-salt textural association that, due to its long lack of permeability (‘locked up in halite’), likely preserves the chemical composition of the original depositional setting (e.g. Zambito et al.). The other school focuses on pore fluid hydrochemistry in subsurface waters, generally using water samples in boreholes, collected from pores and fractures in a carbonate, sandstone or shale host. The nature of fluids in these non-salt sediments, some which have been permeable since deposition, mean fluids experienced ongoing re-supply via crossflow and rock-fluid interaction as the ambient temperature, pressure and salinities evolved in the burial environment. This process shutdown once matrix permeability was lost (Warren et al., 2014).
In the next article, we shall expand our discussion of the significance of CaCl2 brines with a close look at where and how particular calcium chloride minerals can precipitate and be preserved and why some types of calcium chloride salts are more common in particular evaporitic settings.
Drever, J. I., 1997, The geochemistry of natural waters: Surface and groundwater environments: New Jersey, Prentice-Hall Inc., p. 327-351.
Garrett, D. E., 2004, Handbook of Lithium and atural Calcium Chloride; Their deposits, processing, uses and properties Amsterdam, Elsevier Academic Press, 476 p.
Hanor, J. S., 1994, Origin of saline fluids in sedimentary basins: Geological Society, London, Special Publications, v. 78, p. 151-174.
Hanor, J. S., and J. C. McIntosh, 2006, Are secular variations in seawater chemistry reflected in the compositions of basinal brines?: Journal of Geochemical Exploration, v. 89, p. 153-156.
Hardie, L. A., 1990, The roles of rifting and hydrothermal CaCl2 brines in the origin of potash evaporites: a hypothesis: American Journal of Science, v. 290, p. 43-106.
Hardie, L. A., 1996, Secular variation in seawater chemistry: an explanation for the coupled secular variation in the mineralogies of marine limestones and potash evaporites over the past 600 m.y.: Geology, v. 24, p. 279 - 283.
Holland, H. D., and H. Zimmermann, 2000, The Dolomite Problem Revisited: Int. Geol. Rev., v. 42, p. 481-490.
Martini, A. M., 1979, Hydrogeochemistry of Saline Fluids and Associated Water and Gas, Michigan Basin: Doctoral thesis, University of Michigan, 236 p.
McIntosh, J. C., G. Garven, and J. S. Hanor, 2011, Impacts of Pleistocene glaciation on large-scale groundwater flow and salinity in the Michigan Basin: Geofluids, v. 11, p. 18-33.
Warren, J., C. Morley, T. Charoentitirat, I. Cartwright, P. Ampaiwan, P. Khositchaisri, M. Mirzaloo, and J. Yingyuen, 2014, Structural and fluid evolution of Saraburi Group sedimentary carbonates, central Thailand: A tectonically driven fluid system: Marine and Petroleum Geology, v. 55, p. 100-121.
Warren, J. K., 2015, Seawater chemistry (1 of 2): Potash bitterns and Phanerozoic marine brine evolution, Salty Matters blog, www.saltworkconsultants.com.
Wilson, T. P., and T. A. Hewett, 1992, Geochemistry and isotope chemistry of Michigan Basin brines: Devonian formations: Applied Geochemistry, v. 7, p. 81-100.

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