Colicinogenic Plasmid Research Articles

Shigella sonnei utilises colicins during inter-bacterial competition.

The mammalian colon is one of the most densely populated habitats currently recognised, with 1011-1013 commensal bacteria per gram of colonic contents. Enteric pathogens must compete with the resident intestinal microbiota to cause infection. Among these enteric pathogens are Shigella species which cause approximately 125 million infections annually, of which over 90 % are caused by Shigella flexneri and Shigella sonnei. Shigella sonnei was previously reported to use a Type VI Secretion System (T6SS) to outcompete E. coli and S. flexneri in in vitro and in vivo experiments. S. sonnei strains have also been reported to harbour colicinogenic plasmids, which are an alternative anti-bacterial mechanism that could provide a competitive advantage against the intestinal microbiota. We sought to determine the contribution of both T6SS and colicins to the anti-bacterial killing activity of S. sonnei. We reveal that whilst the T6SS operon is present in S. sonnei, there is evidence of functional degradation of the system through SNPs, indels and IS within key components of the system. We created strains with synthetically inducible T6SS operons but were still unable to demonstrate anti-bacterial activity of the T6SS. We demonstrate that the anti-bacterial activity observed in our in vitro assays was due to colicin activity. We show that S. sonnei no longer displayed anti-bacterial activity against bacteria that were resistant to colicins, and removal of the colicin plasmid from S. sonnei abrogated anti-bacterial activity of S. sonnei. We propose that the anti-bacterial activity demonstrated by colicins may be sufficient for niche competition by S. sonnei within the gastrointestinal environment.

Carbapenem-Resistant and ESBL-Producing Enterobacterales Emerging in Central Texas.

Carbapenem-resistant Enterobacterales (CRE) are subject to intense global monitoring in an attempt to maintain awareness of prevalent and emerging resistance mechanisms and to inform treatment and infection prevention strategies. CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are not usually examined collectively in regards to their shared pool of resistance determinants. Here, we genetically and phenotypically assess clinical isolates of CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in the growing region of Central Texas, where CRE are emergent and occurrence of non-carbapenemase-producing-CRE (non-CP-CRE) infections is increasing. CRE (n=16) and ESBL-producing Enterobacterales (n=116) isolates were acquired from a regional hospital in Central Texas between December 2018 and January 2020. Isolates were assessed genetically and phenotypically using antibiotic susceptibility testing, targeted PCR, and whole genome sequencing. CRE infections are increasing in incidence in Central Texas, and Klebsiella pneumoniae is causing the majority of these infections. Moreover, K. pneumoniae sequence type (ST) 307 is commonly found among both non-CP-CRE and EBSL-producing strains. Isolates carry similar plasmids harboring the gene for the ESBL CTX-M-15 and belong to the global lineage, rather than the Texas lineage, of ST307. Antibiotic resistance profiles, sequence data, and clinical records suggest that porin mutations may promote the transition of ST307 isolates from ESBL-producing to non-CP-CRE. In addition to antibiotic resistance mechanisms, several CRE isolates harbor active colicinogenic plasmids, which might influence the competitiveness of these bacteria during patient colonization. K. pneumoniae of the global ST307 lineage is circulating in Central Texas and is responsible for both non-CP CRE and ESBL-producing Enterobacterales infections. Enhanced surveillance is needed to understand the possible routes for the emergence of non-CP-CRE from EBSL-producing strains.

RtcB2-PrfH Operon Protects E. coli ATCC25922 Strain from Colicin E3 Toxin.

In the bid to survive and thrive in an environmental setting, bacterial species constantly interact and compete for resources and space in the microbial ecosystem. Thus, they have adapted to use various antibiotics and toxins to fight their rivals. Simultaneously, they have evolved an ability to withstand weapons that are directed against them. Several bacteria harbor colicinogenic plasmids which encode toxins that impair the translational apparatus. One of them, colicin E3 ribotoxin, mediates cleavage of the 16S rRNA in the decoding center of the ribosome. In order to thrive upon deployment of such ribotoxins, competing bacteria may have evolved counter-conflict mechanisms to prevent their demise. A recent study demonstrated the role of PrfH and the RtcB2 module in rescuing a damaged ribosome and the subsequent re-ligation of the cleaved 16S rRNA by colicin E3 in vitro. The rtcB2-prfH genes coexist as gene neighbors in an operon that is sporadically spread among different bacteria. In the current study, we report that the RtcB2-PrfH module confers resistance to colicin E3 toxicity in E. coli ATCC25922 cells in vivo. We demonstrated that the viability of E. coli ATCC25922 strain that is devoid of rtcB2 and prfH genes is impaired upon action of colicin E3, in contrast to the parental strain which has intact rtcB2 and prfH genes. Complementation of the rtcB2 and prfH gene knockout with a high copy number-plasmid (encoding either rtcB2 alone or both rtcB2-prfH operon) restored resistance to colicin E3. These results highlight a counter-conflict system that may have evolved to thwart colicin E3 activity.

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins.

We have developed the CL7/Im7 protein purification system to achievehigh-yield, high-purity and high-activity (HHH) products in one step. The system is based on the natural ultrahigh-affinity complex between the two small proteins encoded by colicinogenic plasmids carried by certain E. coli strains, the DNAse domain of colicin E7 (CE7; MW~15kDa) and its natural endogenous inhibitor, the immunity protein 7 (Im7; MW~10kDa). CL7 is an engineered variant of CE7, in which the toxic DNA-binding and catalytic activities have been eliminated while retaining the high affinity to Im7. CL7 is used as a protein tag, while Im7 is covalently attached to agarose beads. To make the CL7/Im7 technique easy to use, we have designed a set of the E. coli expression vectors for fusion of a target protein to the protease-cleavableCL7-tag either at the N- or the C-terminus, and also have the options of the dual (CL7/His8) tag. A subset of vectors is dedicated for cloning membrane and multisubunit proteins. The CL7/Im7 system has severalnotable advatantages over other available affinity purification techniques. First, high concentrations of the small Im7 protein are coupled to the beads resulting in the high column capacities (up to 60mg/mL). Second, an exceptional stability of Im7 allows for multiple (100+) regeneration cycles with no loss of binding capacities. Third, the CL7-tag improves protein expression levels, solubility and, in some cases, assists folding of the target proteins. Fourth, the on-column proteolytic elution produces purified proteins with few or no extra amino acid residues. Finally, the CL7/Im7 affinity is largely insensitive to high salt concentrations. For many target proteins, loading the bacterial lysates on the Im7 column in high salt is a key to high purity. Altogether, these properties of the CL7/Im7 system allow for a one-step HHH purification of most challenging, biologically and clinically significant proteins.

Deciphering the Role of Colicins during Colonization of the Mammalian Gut by Commensal E. coli.

Colicins are specific and potent toxins produced by Enterobacteriaceae that result in the rapid elimination of sensitive cells. Colicin production is commonly found throughout microbial populations, suggesting its potential importance for bacterial survival in complex microbial environments. Nonetheless, as colicin biology has been predominately studied using synthetic models, it remains unclear how colicin production contributes to survival and fitness of a colicin-producing commensal strain in a natural environment. To address this gap, we took advantage of MP1, an E. coli strain that harbors a colicinogenic plasmid and is a natural colonizer of the murine gut. Using this model, we validated that MP1 is competent for colicin production and then directly interrogated the importance of colicin production and immunity for MP1 survival in the murine gut. We showed that colicin production is dispensable for sustained colonization in the unperturbed gut. A strain lacking colicin production or immunity shows minimal fitness defects and can resist displacement by colicin producers. This report extends our understanding of the role that colicin production may play for E. coli during gut colonization and suggests that colicin production is not essential for a commensal to persist in its physiologic niche in the absence of exogenous challenges.

Porcine pathogenic Escherichia coli strains differ from human fecal strains in occurrence of bacteriocin types

Enterotoxigenic and Shiga-toxigenic Escherichia coli (i.e., ETEC and STEC) are important causative agents of human and animal diseases. In humans, infections range from mild diarrhea to severe life-threating conditions, while infections of piglets result in lower weight gain and higher pig mortality with the accompanying significant economic losses.In this study, frequencies of four phylogenetic groups, fourteen virulence- and thirty bacteriocin determinants were analyzed in a set of 443 fecal E. coli isolates from diseased pigs and compared to a previously characterized set of 1283 human fecal E. coli isolates collected in the same geographical region. In addition, these characteristics were compared among ETEC, STEC, and non-toxigenic porcine E. coli isolates.Phylogenetic group A was prevalent among porcine pathogenic E. coli isolates, whereas the frequency of phylogroup B2, adhesion/invasion (fimA, pap, sfa, afaI, ial, ipaH, and pCVD432) and iron acquisition (aer and iucC) determinants were less frequent compared to human fecal isolates.Additionally, porcine isolates differed from human isolates relative to the spectrum of produced bacteriocins. While human fecal isolates encoded colicins and microcins with a similar prevalence, porcine pathogenic E. coli isolates produced predominantly colicins (94% of isolates); especially colicins B (42.6%), M (40.1%), and Ib (34.0%), which are encoded on large conjugative plasmids. The observed high prevalence of these colicin determinants suggests the importance of large colicinogenic plasmids and/or the importance of colicin production in intestinal inflammatory conditions.

Enhanced Biofilm Formation and Membrane Vesicle Release by Escherichia coli Expressing a Commonly Occurring Plasmid Gene, kil.

Escherichia coli is one of the most prevalent microorganisms forming biofilms on indwelling medical devices, as well as a representative model to study the biology and ecology of biofilms. Here, we report that a small plasmid gene, kil, enhances biofilm formation of E. coli. The kil gene is widely conserved among naturally occurring colicinogenic plasmids such as ColE1 plasmid, and is also present in some plasmid derivatives used as cloning vectors. First, we found that overexpression of the kil gene product dramatically increased biofilm mass enriched with extracellular DNA in the outer membrane-compromised strain RN102, a deep rough LPS mutant E. coli K-12 derivative. We also found that the kil-enhanced biofilm formation was further promoted by addition of physiologically relevant concentrations of Mg2+, not only in the case of RN102, but also with the parental strain BW25113, which retains intact core-oligosaccharide LPS. Biofilm formation by kil-expressing BW25113 strain (BW25113 kil+) was significantly inhibited by protease but not DNase I. In addition, a large amount of proteinous materials were released from the BW25113 kil+ cells. These materials contained soluble cytoplasmic and periplasmic proteins, and insoluble membrane vesicles (MVs). The kil-induced MVs were composed of not only outer membrane/periplasmic proteins, but also inner membrane/cytoplasmic proteins, indicating that MVs from both of the outer and inner membranes could be released into the extracellular milieu. Subcellular fractionation analysis revealed that the Kil proteins translocated to both the outer and inner membranes in whole cells of BW25113 kil+. Furthermore, the BW25113 kil+ showed not only reduced viability in the stationary growth phase, but also increased susceptibility to killing by predator bacteria, Vibrio cholerae expressing the type VI secretion system, despite no obvious change in morphology and physiology of the bacterial membrane under regular culture conditions. Taken together, our findings suggest that there is risk of increasing biofilm formation and spreading of numerous MVs releasing various cellular components due to kil gene expression. From another point of view, our findings could also offer efficient MV production strategies using a conditional kil vector in biotechnological applications.

Characterization of E-type colicinogenic plasmids from Shigella sonnei.

Colicinogenic plasmids encode toxic proteins which have antagonistic activity against closely related bacteria. This study describes the molecular characterization of three colicinogenic plasmids designated as pSSE3, pSSE and pSSE2, each with a molecular size of ∼6 kb, identified in clinical isolates of Shigella sonnei. Sequence analysis revealed that pSSE and pSSE2 shared extensive sequence homology with each other and with Escherichia coli E-type colicinogenic plasmids. The plasmid pSSE3 lacked an additional gene imparting immunity to colicin E8, a unique feature not observed in any of the previously reported sequences of colicin E3 plasmids. Incomplete digestion of colicinogenic plasmids by restriction endonucleases, metachromatic staining with acridine orange and presence of single stranded initiation (ssi) region confirmed the coexistence of ssDNA along with dsDNA. Plasmid copy number as determined by real-time PCR was found to be about 20. Transmission electron microscopy revealed DNA impairment in test bacteria after colicin exposure. We hypothesize that S. sonnei has acquired E-group colicin plasmids from its close relative E. coli, with their sequences undergoing subtle changes depending on the cohabitation in the same milieu.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has “chosen” highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili ( pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

Complete genome sequence of the incompatibility group I1 plasmid R64

A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously ( Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826 bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn 10 and Tn 6082, respectively. These transposons and two insertion elements, IS 2 and IS 1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure.

Colicin biology.

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

Characterization of two cryptic Helicobacter pylori plasmids: a putative source for horizontal gene transfer and gene shuffling.

Many Helicobacter pylori isolates carry cryptic plasmids of extremely variable size. In this study we analyzed two H. pylori plasmids, pHel4 and pHel5, from H. pylori strains P8 and P29, respectively. Plasmid pHel4 consists of 10,970 bp, constituting 15 putative open reading frames (ORFs), whereas pHel5 consists of 18,291 bp, constituting 17 ORFs. The findings that both plasmids encode a conserved RepA protein and that both have an origin of replication containing an iteron place them in the group of theta plasmids. In pHel4, the products of the overlapping orf4C, orf4D, orf4E, and orf4F sequences are homologous to MobA, MobB, MobC, and MobD, encoded by colicinogenic plasmids, suggesting that pHel4 might be mobilizable. A further putative operon consists of orf4B and orf4A, the products of which are homologous to microcin C7 (MccC7) biosynthesis and secretion proteins MccB and MccC, respectively. Plasmid pHel5 carries putative genes encoding proteins with homology to an endonuclease and gene products of an H. pylori chromosomal plasticity zone. Both plasmids contain repeat sequences, such as the previously identified R2 repeat, which are considered preferred recombination sites. In pHel4, a new repeat sequence (R4 repeat), which seems to act as a hot spot for site-specific recombination, was identified. All H. pylori plasmids characterized so far have a modular structure. We suggest a model that explains the existing plasmids by insertions and deletions of genetic elements at the repeat sequences. A genetic exchange between plasmids and the bacterial chromosome, combined with plasmid mobilization, might add a novel mechanism to explain the high genetic macrodiversity within the H. pylori population.

How bacteria protect themselves against channel-forming colicins.

Here we review the mechanisms that bacterial cells use to protect themselves against channel-forming colicins. Four mechanisms are examined: immunity, resistance, tolerance and PacB character. Immunity confers protection to colicinogenic cells against the colicin they produce, since the colicinogenic plasmid bears the genetic determinant for such immunity protein. Resistance is provided by modifications on colicin receptors located on the outer membrane. It prevents colicin adsorption and protects against those colicins sharing a common receptor. Tolerance is achieved by changes in the translocation system. The adsorbed colicin is not translocated toward the periplasmic space. This impedes its insertion into the cell membrane as well as the formation of the transmembrane channel. Tolerance confers protection against colicins that share the same translocation system. Finally, we discuss the PacB character, that confers protection against all known channel-forming colicins. The latter property is encoded by non-colicinogenic plasmids in the H-incompatibility complex.

Structure and function of plasmid pColD157 of enterohemorrhagic Escherichia coli O157 and its distribution among strains from patients with diarrhea and hemolytic-uremic syndrome.

In this study, pColD157, a 6.7-kb colicinogenic plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL40cu, was characterized by restriction mapping and determination of its complete nucleotide sequence. The sequence consists of 6,675 bp and shows a high degree of similarity to the nucleotide sequence of colicinogenic plasmids pColD-CA23 and pColK. Seven potential genes were located on pColD157, three of which were closely related (>97.9%) to the colicin D structural gene and the corresponding immunity and lysis genes of plasmid pColD-CA23, and these were therefore designated cda, cdi, and cdl, respectively, using the reference extension -CL40 for differentiation. The adjacent 3' region is related to the origin of replication of pColD-CA23. In contrast, the remaining part of the plasmid harbors a cluster of genes, closely related to the mobilization genes of pColK, which is followed by a 0.3-kb stretch homologous to the pColK resolution function. These determinants were designated mbdA, mbdB, mbdC, and mbdD and cdr, respectively. Southern blot analysis was performed with a probe specific for the cda gene of pColD157 and two groups of EHEC O157:H7 isolates from patients with diarrhea or hemolytic-uremic syndrome resident in Germany. Whereas 16 of 46 E. coli O157 strains isolated between 1987 and 1991 harbored plasmid pColD157, only 1 of 50 strains isolated during 1996 carried this plasmid. In addition, all strains harboring plasmid pColD157 were shown to have colicinogenic activity.

The association between colicinogenicity and pathogenesis among uropathogenic isolates of Escherichia coli

Colicins have previously been thought to play an indirect role in bacterial pathogenesis. We describe here an association between colicinogenicity and pathogenesis among uropathogenic E. colistrains based on 568 clinical isolates. Significantly more strains isolated from patients with the symptomatic infections pyelonephritis and cystitis produced colicin, 44.4% ( P<0.05) and 51.5% ( P<0.01), respectively, than those strains isolated from patients with asymptomatic infections (32.5%). Attempts to identify new colicins produced by the 232 colicin +isolates showed that 57.3% did not belong to any known colicin type. The colicin V genotype was found in only 10.8% of the colicin +isolates, suggesting the possibility of different colicinogenic plasmids predominating during urinary tract infections.

Genetic Organization and Evolution of the Cryptic Plasmid of Neisseria gonorrhoeae

Three promoters have been identified on the phenotypically cryptic plasmid of Neisseria gonorrhoeae that can direct transcription in Escherichia coli. These promoters do not seem to function at a detectable level when N. gonorrhoeae is grown in vitro under normal growth conditions. The T7 RNA polymerase expression system has been used to identify the proteins encoded by this plasmid, and a series of subclones were constructed and used to localize the gene encoding each protein. In a search of the sequence databases, we have discovered that the derived amino acid sequence of a 1.2-kb segment of the plasmid shows a high degree of sequence similarity to the Mob proteins encoded by the colicinogenic plasmids ColA, ColE1, and ColK of E coli. The 1.2-kb segment contains part of a Col mobilization region that has subsequently been inactivated by a small duplication. During the recombination event that formed the cryptic plasmid one of the mobilization genes was fused to a cryptic plasmid open reading frame to form the cppB gene. Despite the manner in which it was formed, we have shown that the cppB gene can be expressed when N. gonorrhoeae is grown under the appropriate environmental conditions.

Physical and functional characterization of the ColS8 plasmid

The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported. Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid. Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined. The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids. There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions. In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein. One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions. Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations.

Par site of the ColN plasmid: Structural and functional organization

A par site involved in the resolution of multimeric plasmid DNA forms was localized in a 679 bp SalI-KpnI fragment of the small colicinogenic plasmid ColN. It was shown that replication of the monomeric pUC19 recombinant plasmid carrying the par region of ColN does not result in the formation of significant numbers of multimers. In order to function properly, the ColN multimer resolution mechanism requires the product of the xerA gene, just as in the case of ColE1. Nucleotide sequence analysis of the par region of ColN revealed substantial homology with the par locus of the ColE1 plasmid. The results of this study and data from the literature indicate that the par sites of ColE1-type plasmids have substantial homology and the same mechanism of action, and in fact represent a universal stability module for small multicopy colicinogenic plasmids.