Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids.

The transmission of plasmid pCU1 (or other IncN group plasmid) into a population of Klebsiella oxytoca cells reduces the viability of the population. A 2,400-bp region adjacent to traA is responsible for this phenotype and includes two regions, called kikA and kikC. Klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. To obtain insight into the mode of this apparent lethality, an appropriate pCU1lacZ derivative was constructed. It could be introduced with high efficiency into Klebsiella cells. Analyses of the resultant colonies indicate that the loss of viability is not a consequence of the death of plasmid-free segregants. On the contrary and unlike postsegregational killing by plasmids, cells survived by losing the plasmid or by acquiring, secondarily, a chromosomal mutation which confers immunity to killing.     

Regions on plasmid pCU1 required for the killing of Klebsiella pneumoniae.

Plasmid pCU1 was Kik+ (promotes killing of Klebsiella pneumoniae). All Tn5 insertions within the tra region of pCU1 were Kik-. Two other regions, kikA and kikB, were needed. They may be separated on different plasmids, but both must be mobilized into Klebsiella pneumoniae. Establishment of one kik region in K. pneumoniae followed by receipt of the second did not lead to killing. Kik was therefore intracellular and required concerted and transient action of both regions.     

Conditionally lethal genes in the N pilus region of plasmid pCU1.

Plasmid genes or regions that are conditionally lethal to Escherichia coli have been called kil and those lethal to Klebsiella but not to E. coli have been called kik. Both classes of genes are found in or close to the N pilus region of the plasmid pCU1 and the closely related plasmid pKM101. Here we describe two new and overlapping lethal genes that are located between kikA and traA of the plasmid pCU1 and display host specificity. KilC is lethal in E. coli and Klebsiella while kikC is lethal only in Klebsiella. The previously identified korA gene is sufficient to override the lethality of kilC in trans or in cis but is insufficient to override kikC. kilC expression in E. coli leads to cell death accompanied by an increase in average cell length without affecting septum formation.     

A demonstration that pCU1 tra gene products are not required in the killing of Klebsiella pneumoniae

IncN group plasmids, including pCU1, are able to kill Klebsiella pneumoniae when conjugatively transferred from an Escherichia coli donor. Transposon mutagenesis and deletion analysis of the known tra complementation groups were used to demonstrate that the tra gene products inactivated are not required for the Kik phenotype.     

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli     

Sequencing, cloning and expression of a β-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4

Abstract We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is qonserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.     

Analysis of the replication region of a mycobacterial plasmid, pMSC262.

We determined the nucleotide sequence of a DNA fragment which contains the replication region of pMSC262, a Mycobacterium scrofulaceum plasmid used to construct the Mycobacterium-Escherichia coli shuttle vector. The complete sequence of the fragment contained 2,504 bp with an overall G+C content of 69.8%. By deletion analysis, we found that the minimum length required for plasmid replication in M. bovis BCG was about 1.6 kb. Within this region, several open reading frames (ORFs) and a putative replication origin (ori) were identified by computer analysis. One of the ORFs, ORF2, which encodes a putative 28.9-kDa basic protein with characteristics of DNA-binding proteins, appeared to be involved in replication of the plasmid in BCG. By separation of ORF2 and the putative ori region, it was revealed that the relative locations of ORF2 and the putative ori region are likely important for replication in BCG. No DNA or amino acid homologies were found between this replication region and that of pAL5000, another mycobacterial plasmid used for vector plasmid construction. In addition, we found that this replicon did not lead to replication in E. coli and was compatible in BCG with pAL5000-derived vector plasmid pYUB75 (R. G. Barletta, D. D. Kim, S. B. Snapper, B. R. Bloom, and W. R. Jacobs, J., J. Gen. Microbiol. 138:23-30, 1992).     

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.     

Analysis of an Escherichia coli mutant strain resistant to the cell-killing function encoded by the gef gene family

The chromosomal genes gef and relF from Escherichia coli and the plasmid-encoded genes hok, flmA, srnB, and pndA constitute the gef gene family, which encodes a cell-killing function. In order to investigate the mechanism of cell killing we have isolated an E. coli mutant strain that is resistant to the overexpression of the toxic proteins encoded by the gef gene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an operon.     

Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum

Plasmid pAM5 of Acidiphilium multivorum JCM-8867 has been completely sequenced by initial cloning of HindIII-PstI fragments followed by primer walking. It has a size of 5161bp and single site for several restriction enzymes as revealed by DNA sequencing. Sequence analysis predicts five putative open reading frames. ORF1 and ORF3 show significant identity with various plasmid encoded mobilization (Mob) and replication initiation (Rep) proteins, respectively. The putative Mob protein has several characteristics of the MOB(Q) family having the motifs with conserved amino acid residues. Upstream of the Mob ORF, there exists a 34bp oriT region having a nic consensus sequence. The constructed plasmid pSK1 bearing pAM5 mob region can be mobilized to Escherichia coli in presence of conjugative plasmid pRK2013. The replication module comprises of several DnaA like boxes, several perfect direct and inverted repeats, a potential prokaryotic promoter and putative rep gene. The rep module is very similar to several theta replicating iteron family plasmids, suggesting pAM5 replication to follow the same course. Any phenotypic character determinant (e.g., metal resistance, antibiotic resistance etc.) gene is absent in pAM5, suggesting this plasmid to be cryptic in nature. However, a pAM5 derivative plasmid named pSK2, containing the putative pAM5 rep region, can replicate and be stably maintained in Acidiphilium, Acidocella, and E. coli strains; it can also carry foreign DNA fragments. Thus, pSK2 could serve as a cloning shuttle vector between these bacteria. It was observed that pAM5 Rep is essential for pSK2 to replicate in acidophiles. In its natural host, A. multivorum JCM-8867, pAM5 maintains a copy number of 50-60, and its derivative pSK2 maintains a comparatively, higher copy number in E. coli than in acidophiles.     

Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale

Plasmid pCR1 is a cryptic plasmid harboured by Corynebacterium renale. It is the smallest corynebacterial plasmid known to date. Although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. It can also replicate in Escherichia coli, in which it is stably maintained. The copy number of pCR1 in this host is higher than that of pUC19, with which it shows unidirectional incompatibility. It is also incompatible with pBK2, a plasmid bearing the common corynebacterial replicon pBL1. Its size is 1488bp, as revealed by DNA sequencing. A total of eight open reading frames (ORF) were detected in this plasmid, the largest of which codes for a putative Rep protein of predicted molecular mass of 21kDa. The plasmid pCR1 can be mobilized by the plasmid R6K from E. coli to other corynebacteria. Sequence analysis revealed the presence of an oriT homologous to that of R64. An E. coli plasmid pKL1 shows more than 90% identity with pCR1. Like many coryenbacterial plasmids, pCR1 also replicates by rolling circle mode.     

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.     

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region (tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.     

Molecular characterization of a gene encoding a photolyase from Streptomyces griseus.

By using a synthetic DNA probe derived from an amino acid sequence in the most conserved region of three known photolyases (Escherichia coli, Anacystis nidulans and Saccharomyces cerevisiae), we isolated a DNA fragment containing two long open reading frames (ORFs) from a genomic DNA library of Streptomyces griseus. One ORF encodes a polypeptide of 455 amino acids (Mr 50594), which exhibits substantial similarities with the other three photolyases. Photoreactivation-repair deficient E. coli cells could be converted into photoreactivatable ones by introduction of plasmids harboring this ORF, indicating that this is the photolyase gene of S. griseus. The deduced aa sequence of Streptomyces photolyase was most similar to that of E. coli. The putative DNA binding site as well as cofactor binding regions were proposed.     

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.