Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum ␤-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla CTX-M-3 , it also bears the bla TEM-1 , aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of ␤-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla CTX-M-3 , accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

a b s t r a c tSystems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here. Crown

Plasmids classified to the IncP-1 incompatibility group belong to the most stably maintained mobile elements among low copy number plasmids known to date. The remarkable persistence is achieved by various tightly controlled stability mechanisms like active partitioning, efficient conjugative transfer system, killing of plasmid-free segregants and multimer resolution. The unique feature of IncP-1 plasmids is the central control operon coding for global regulators which control the expression of genes involved in vegetative replication, stable maintenance and conjugative transfer. The multivalent regulatory network provides means for coordinated expression of all plasmid functions. The current state of knowledge about two fully sequenced plasmids RK2 and R751, representatives of the IncP-1alpha and IncP-1beta subgroups, is presented.

The kfrA gene of the IncP-1 broad-host-range plasmids is the best-studied member of a growing gene family that shows strong linkage to the minimal replicon of many low-copy-number plasmids. KfrA is a DNA binding protein with a long, alpha-helical, coiled-coil tail. Studying IncP-1b plasmid R751, evidence is presented that kfrA and its downstream genes upf54.8 and upf54.4 were organized in a tricistronic operon (renamed here kfrA kfrB kfrC), expressed from autoregulated kfrAp, that was also repressed by KorA and KorB. KfrA, KfrB and KfrC interacted and may have formed a multi-protein complex. Inactivation of either kfrA or kfrB in R751 resulted in long-term accumulation of plasmid-negative bacteria, whereas wild-type R751 itself persisted without selection. Immunofluorescence studies showed that KfrA R751 formed plasmid-associated foci, and deletion of the C terminus of KfrA caused plasmid R751DC 2 kfrA foci to disperse and mislocalize. Thus, the KfrABC complex may be an important component in the organization and control of the plasmid clusters that seem to form the segregating unit in bacterial cells. The studied operon is therefore part of the set of functions needed for R751 to function as an efficient vehicle for maintenance and spread of genes in Gram-negative bacteria. INTRODUCTIONSequencing of bacterial genomes has revealed not only that some chromosomes are very similar to megaplasmids in their replication and stable inheritance mechanisms, but also that most standard oriC-based chromosomes carry a range of stable inheritance functions, already studied in some detail on bacterial plasmids (Gerdes et al., 2000a). This includes active partitioning systems of the parAB family (Gerdes et al., 2000b;Bignell & Thomas, 2001), multimer resolution systems of the xerC/D family (Summers & Sherratt, 1984; Blakely et al., 1993) and a variety of postsegregational killing systems (Gerdes et al., 2000a;Zielenkiewicz & Ceglowski, 2001). Thus, studies on bacterial plasmids, particularly large, low-copy-number plasmids, can be of direct relevance for understanding key events of the bacterial cell cycle. Genomic approaches also reveal genes that are conserved between many systems and cluster with known replication and stable inheritance functions, but do not yet have a role assigned. One such gene family has as its archetype the kfrA gene of IncP-1a plasmid RK2 that we studied many years ago, but for which we did not establish a phenotype, although we speculated that it may be involved in active partitioning (Jagura-Burdzy & Thomas, 1992;Williams & Thomas, 1992). KfrA RK2 is 308 aa in length, is a site-specific DNA binding protein, and is predicted to be almost 100 % a-helical, which is consistent with its circular dichroism (CD) spectrum (Jagura-Burdzy & Thomas, 1992). It is predicted to consist of an N-terminal, globular, DNA binding domain and an extended coiled-coil tail. Database searching revealed similarity to proteins of the myosin/ kinesin family and, recently, to prokaryotic structural maintenace of chromosomes (...

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