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DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism.

The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol Oxidoreductases↗

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.

Ampicillin↗

Unexpected divergence and molecular coevolution in yeast plasmids.

Four closely related species of yeast possess multicopy nuclear plasmids whose shared molecular architecture demonstrates a common ancestor, despite their lack of discernible DNA sequence homology. Each plasmid encodes three proteins which have equivalent essential functions in plasmid maintenance. These three groups of proteins show markedly different degrees of conservation, so that although we have successfully aligned sequences for two groups, members of the third group have diverged to such an extent that they cannot be aligned. All the proteins are sufficiently different that they function only in conjunction with their encoding plasmid. These proteins have therefore conserved their functional interactions with the relevant DNA sequences of their particular plasmids, despite lack of amino acid sequence conservation. The maintenance of function in the face of DNA sequence divergence is analogous to the coevolution of ribosomal DNA promoters and RNA polymerase I, and suggests that molecular drive may be an important force in the evolution of these plasmids. This view is reinforced by the inconsistent phylogenetic relationships determined from the two alignment sets, and by the contradiction that the two plasmids known to be the closest related taxonomically and by their host interchangeability are suggested to be the most distant by their sequences.

Amino Acid Sequence↗

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae.

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.

Base Sequence↗

The relationship of the delta transfer factor and KColIb to the ColIb plasmid.

The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared. Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids. The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid. It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments. However, ColIb, KColIb and delta were homologous for at least 70% of their lengths. The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb. In addition, delta and KColIb differed from ColIb at similar sites. The possible evolution of these plasmids is discussed.

Bacteriocin Plasmids↗

Mathematics of microbial plasmid instability and subsequent differential growth of plasmid-free and plasmid-containing cells, relevant to the analysis of experimental colony number data.

Differential growth of plasmid-containing and plasmid-free microbial cells occurs in many and probably most plasmid systems. Misinterpretation of differential growth as replicational, recombinational, or segregational stability or instability can unfortunately result in grossly erroneous conclusions about replication, recombination, or segregation in plasmid model systems for studies of such phenomena. The differential growth rate should ideally be measured every time that the rate of loss due to instability per se is measured. Unfortunately, the possibility of differential growth has been ignored in most plasmid model systems, since the mathematics of instability and differential growth has not usually been dealt with in ways that are intuitively understandable to experimental microbiologists. Nevertheless, rapid diagnosis of differential growth, and accurate estimation of differential growth rate and rate of loss due to instability per se, can be done by analysis of colony number data using only the relatively simple mathematics described in this review. This review is intended for experimental microbiologists rather than for theoretical population geneticists or for pure mathematicians. However, the same mathematics described in this review is also applicable to certain simple model systems for plasmid ecology or evolution in natural or clinical environments.

Bacteria↗

The evolution of pTF-FC2 and pTC-F14, two related plasmids of the IncQ-family.

Two plasmids, pTF-FC2 and pTC-F14, that belong to the IncQ-like plasmid family were isolated from two related bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, respectively. The backbone regions of the two plasmids share a sufficiently high amount of homology to indicate that they must have originated from the same ancestral plasmid. Although some of their replication proteins could complement each other, the plasmids have evolved sufficiently for their replicons to have become compatible. This compatibility has occurred by changes in the iteron sequence, RepC (iteron binding protein) specificity and the regulation properties of the RepB primase. Two of the five mobilization genes have remained highly conserved, whereas the other three genes appear to have evolved such that each plasmid is mobilized most efficiently by a different self-transmissible plasmid. Plasmids pTF-FC2 and pTC-F14 do not appear to compete at the level of mobilization. The antitoxins of the toxin-antitoxin (TA) plasmid stability systems were partly able to neutralize the toxins of the other plasmid and also to partly cross-regulate the TA systems of the other plasmid with the antitoxin of pTF-FC2 being the most effective cross-regulator. Other aspects of the evolution of the two plasmids are described and the danger of making the assumption that incompatibly of IncQ-like plasmids is a reflection of the degree of relatedness of two plasmids is discussed.

Acidithiobacillus↗

Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, and -32) plasmids.

The spread of CTX-M-1-like enzymes in Spain is associated with particular plasmids of broad-host-range IncN (blaCTX-M-32, blaCTX-M-1), IncL/M (blaCTX-M-1), and IncA/C2 (blaCTX-M-3) or narrow-host-range IncFII (blaCTX-M-15). The identical genetic surroundings of blaCTX-M-32 and blaCTX-M-1 and their locations on related 40-kb IncN plasmids indicate the in vivo evolution of this element.

Drug Resistance, Bacterial↗

Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon.

Aphids obtain tryptophan from prokaryotic endosymbionts assigned to the genus Buchnera. The rate-limiting enzyme in tryptophan biosynthes, anthranilate synthase, is encoded by the genes trpE and trpG. In Buchnera within the family Aphididae, trpEG is plasmid-borne, apparently as an adaptation to overproduce tryptophan for hosts. To explore the evolution of these plasmids, sequences for trpEG, the upstream region containing the plasmid origin of replication, and chromosomal trpB were obtained for Buchnera of three species in the aphid genus Uroleucon and analyzed together with sequences for six other aphid species. Phylogenies based on trpB and trpEG agree with each other and with previous views of aphid phylogeny. Synonymous substitutions are about twice as high for plasmid-borne genes as for chromosomal genes in the same lineages, suggesting higher mutation rates for genes on plasmids. Nonsynonymous rates for trpEG are accelerated within Buchnera of Uroleucon, indicating a change in selection intensity within this genus. Accelerated evolution within Uroleucon also seems to characterize the upstream region containing the putative origin of replication.

Animals↗

The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in Rhizobiaceae.

The Ri (root-inducing) plasmid in Agrobacterium rhizogenes and Ti (tumor-inducing) plasmid in Agrobacterium tumefaciens have provided the fundamental basis for the construction of plant vectors and transgenic plants. Recently, the determination of the first complete nucleotide sequence of the Ti plasmid (pTi-SAKURA) has been successful. To understand the general structure of these oncogenic T-DNA transfer plasmids, the whole nucleotide sequence of a mikimopine-type Ri plasmid, pRi1724, was analyzed. The plasmid is 217,594 bp in size, and has 173 open reading frames (ORFs) in total, which are asymmetrically distributed. Except for 27 ORFs, which are unknown, 173 ORFs were classified into 12 groups as follows: three for DNA replication, nine for plasmid modification, 22 for conjugation, 26 for virulence, 11 for T-DNA gene, 19 for mikimopine/mikimopine-lactam transport, ten for an unknown opine metabolism, seven for transcriptional regulator, five for sugar transport, five for glycerol metabolism, four for chemoreceptor and 32 for others. The elucidated chimeric structure of pRi1724 interestingly indicates that the evolution of Rhizobiaceae plasmids seems to have kept interactions among the plasmids; especially, the genes and elements for a conjugal transfer of pRi1724 had clearly closer kinship to those of a Sym (symbiotic) plasmid, pNGR234a in Rhizobium sp. than those of Ti plasmids. By using sequencing and Northern analysis, we examined the metabolic pathway and gene expression of mikimopine, which is probably an Ri-specific opine.

ATP-Binding Cassette Transporters↗

Cloning yeast telomeres on linear plasmid vectors.

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.

Animals↗

Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids.

The primary DNA processing protein for conjugative mobilization of the plasmid R1162 is the transesterase MobA, which acts at a unique site on the plasmid, the origin of transfer (oriT). Both MobA and oriT are members of a large family of related elements that are widely distributed among bacteria. Each oriT consists of a highly conserved core and an adjacent region that is required for binding by its cognate MobA. The sequence of the adjacent region is important in determining the specificity of the interaction between the Mob protein and the oriT DNA. However, the R1162 MobA is active on the oriT of pSC101, another naturally occurring plasmid. We show here that MobA can recognize oriTs having different sequences in the adjacent region and, with varying frequencies, can cleave these oriTs at the correct position within the core. Along with the structure of the oriTs themselves, these characteristics suggest a model for the evolution of this group of transfer systems.

Bacterial Proteins↗

IS257-mediated cointegration in the evolution of a family of staphylococcal trimethoprim resistance plasmids.

Analyses of the Staphylococcus epidermidis multiresistance plasmids pSK697 and pSK818 have revealed them to be closely related to the trimethoprim resistance plasmid pSK639, also isolated from S. epidermidis. pSK697 and pSK818 were found to contain a cointegrated copy of a second plasmid related to the S. epidermidis multidrug antiseptic and disinfectant resistance plasmid pSK108 and the S. aureus tetracycline resistance plasmid pT181, respectively. In contrast to pSK639, both plasmids were found to contain a third copy of IS257, such that the integrated plasmids in both cases are flanked by a copy of this element. This organization and the presence of duplicated sequences at the extremities of the integrated plasmids implicate IS257 in the formation of these cointegrate plasmids. Sequence analysis of the IS257 elements from these plasmids has provided insights into the probable mechanism of cointegration, viz., nonresolved replicative transposition of IS257.

Amino Acid Sequence↗

Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon.

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that Sfx and H-NS share similar DNA motifs and a preference for negative supercoiling, but occupy reciprocal genomic niches; Sfx is enriched on the R6K vir operon despite weak chromosomal binding, whereas H-NS displays the opposite preference. We show that Sfx binding to vir DNA critically depends on DNA topology and hypothesize that its selective targeting to R6K is mediated by Sfx-vir interactions and phase separation. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable bridged nucleoprotein filaments that are impermeable to competitors such as H-NS. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life.

Operon↗

Plasmid-linked resistance to inorganic salts in Staphylococcus aureus.

The penicillinase plasmids, a series of extrachromosomal resistance factors in Staphylococcus aureus, were found to carry determinants of resistance to a series of inorganic ions as well as resistance to penicillin and, in some cases, erythromycin. Most of the ions involved were inhibitory but not lethal to the bacteria; the resistance markers conferred an increase in resistance by comparison with susceptible organisms of between 3- and 100-fold, depending on the ion involved. Separate genetic loci for resistance to arsenate, arsenite, lead, cadmium, mercuric, and bismuth ions were demonstrated. Resistance to antimony and resistance to zinc were also found but were not separated genetically from resistance to arsenite and cadmium, respectively. The ion resistance markers appeared to form a cluster on the plasmid, with no other known marker within it. Naturally occurring plasmids were observed that lacked one or more of these ion resistance markers, as well as penicillinase-negative strains that were resistant to one or more of the ions. The patterns of markers carried by these various strains may provide some understanding of the evolution of a plasmid linkage group.

Arsenic↗

Phylogenetic analysis of the spirochetes Borrelia parkeri and Borrelia turicatae and the potential for tick-borne relapsing fever in Florida.

Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identities of these spirochetes in the United States. DNA sequences of four chromosomal loci (the 16S rRNA gene, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. The FCB was clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes.

Bacterial Proteins↗

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1.

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.

Chromosome Mapping↗

A new revision of the sequence of plasmid pBR322.

A revised sequence in the region immediately upstream from the rop gene of pBR322 is reported. Two base pairs in the accepted sequence do not exist in the plasmid DNA. Specifically, a TA base pair is missing at sequence coordinate 1893 [Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77-90] and an AT base pair is missing at position 1915, giving a total size for pBR322 of 4361 bp. These changes are in a potential translation initiation sequence and probably reflect errors in the original sequence rather than recent evolution of the plasmid.

Base Composition↗