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

Abstract

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.All conjugative antibiotic resistance plasmids of the incompatibility group N of gram-negative bacteria confer on their hosts a phenotype called Kik ϩ . The phenotype consists of a marked reduction of the viability of Klebsiella oxytoca (previously Klebsiella pneumoniae [30,31,42]) recipients but not of Escherichia coli K-12 recipients following matings with the donor hosts on solid surfaces, a condition that is necessary for the efficient conjugative transfer of the plasmid. The phenotype was shown to have an intracellular basis and was initially called Kil (30,31), but following the use of the designation kil for plasmid loci conditionally lethal in E. coli (6, 41) and to indicate its host specificity, it was renamed Kik (38). Further studies were undertaken with a plasmid of this group called pCU1 that was the subject of detailed studies in our laboratory. It was shown by transposon Tn5 mutagenesis that a locus called kikA had an important role in this phenotype. The locus mapped close to one end of the conjugative transfer (tra) region of the plasmid, but the Tn5 mutants did not alter the efficiency of conjugative transfer between E. coli organisms (38). Hengen et al. (12) cloned and sequenced kikA and constructed a plasmid derivative in which kikA is controlled by the tac promoter. This cloned plasmid could be transformed efficiently into and maintained in Klebsiella cells. Upon induction with isopropyl-␤-D-thiogalactopyranoside (IPTG), the Klebsiella cells lost their viability. This showed that mating was not essential to bring about Klebsiella cell lethality and that loss of viability of Klebsiella cells could be mediated by events that occurred in them. While these observations showed that kikA has an important intracellular role in determining Klebsiella lethality, previous observations (11, 12, 18) have shown that there are loci on pCU1 and the closely related plasmid pKM101 that are lethal to E. coli (kil loci) in the absence or dysfunction of other, cognate loci (kor loci). Conceivably, such loci could also contribute to lethality of pCU1 in Klebsiella cells.The...

Conditionally lethal genes associated with bacterial plasmids

“…coli (Saltman et al, 1991) ; (v) the chromosomal kicB gene has been reported to interfere with protein synthesis (Feng et al, 1994) and a novel killer system has been suggested for plasmid Rtsl (Tian et al., 1996); and (vi) the indication that carriage of pCUl by K . oxytoca results in a specific chromosomal mutation hints at the possible involvement of plasmids in directly modifying the genetic diversity of bacterial hosts (Rodriguez et al, 1995). It is likely that future investigations will identify as yet unknown functions associated with these systems.…”

“…This region was called k i k A and subsequent studies showed that intracellular expression of kikA in K. oxytoca was lethal whilst expression in E. coli was not (Hengen et al, 1992). Two other loci, kikB (Thatte et al, 1985) and kikC (Rodriguez et al, 1995), have also been implicated in reducing the viability of K . oxytoca and remain to be analysed.…”

“…Analysis of lac2 expression from pCUl : lac2 in developing colonies following conjugation suggested that the mutation conferring immunity to the Kik' phenotype occurs late during the development of a bacterial colony. These observations led to the proposal of a hypothesis whereby the expression of K. oxytoca-lethal genes arrests the growth of the recipient, thus allowing for a rare secondary chromosomal mutation which renders the cell immune to killing by kik genes (Rodriguez et al, 1995). The mutation induced in K .…”

“…oxytoca cells that survived mating with pCUl had acquired a chromosomal mutation (kik-immune) that protected them against killing by pCUl in subsequent matings (Rodriguez et al, 1995). Mating experiments using prototrophic E. coli donors carrying pCUl : lac2 with auxotrophic K. oxytoca recipients demonstrated that the plasmid pCU1 is deleterious to the K. oxytoca host when it is present in the cell (Rodriguez et al, 1995). The observed lethality was therefore not a consequence of segregational plasmid loss followed by the killing of plasmid-free cells as has been observed in E. coli with a number of plasmids (see above).…”

Complete Nucleotide Sequences of bla KPC-4 - and bla KPC-5 -Harboring IncN and IncX Plasmids from Klebsiella pneumoniae Strains Isolated in New Jersey

“…pBK31551 also possesses two modules that are predicted to be involved in the conjugative transfer of plasmid DNA. The first module consists of 11 genes (traL, traM, traA, traB, traC, traD, traN, traE, traO, traF, and traG) flanked by a group of plasmid core genes involved in regulating transfer and replication (korA and korB) (39), excluding DNA entry (eex) (40), DNA degradation (nuc) (41), and killing of Klebsiella (kikA) (42,43) (Fig. 1).…”