Plasmid Exchange Research Articles

Role of bioinformatics in the development of new antibacterial therapy

Bacterial virulence and pathogenicity has its genesis in plasmid exchange, horizontal gene transfer, transposons carrying virulent genes, genome rearrangement, missing genes causing gaps in pathways, gene mutations altering gene functionality, pathogenicity islands, efflux pumps, pore-inducing proteins causing cell death and release of exotoxins that can harm the host. In the past, docking based on 3D molecular modeling to identify the compounds that will bind to a protein disrupting the pathway has been used for antibiotic development. However, current hospital practice aided by fast mutation of virulent genes has produced many drug-resistant strains capable of surviving extreme stress conditions caused by antibiotics. Recent bioinformatics research can help remedy the situation by helping to understand the exact functionality of the biomachine inside the bacteria and the host pathogen interaction at the level of individual pathogens, and can reduce the drug-development time in future.

Conjugation-mediated plasmid exchange between bacteria grown under space flight conditions

Mobile genetic elements (MGE) such as phages, plasmids and transposons play a crucial role in bacterial adaptation and evolution. These MGE mobilize and reorganize genes within a given genome or between bacterial cells. The impact of space flight conditions on these processes is largely unknown. The Mobilisatsia/Plasmida experiment was set up to investigate the impact of space flight conditions on plasmid-mediated conjugation. The experiment was done aboard the International Space Station during the Soyuz Mission 8S (April 19(th) until April 30(th) 2004). An experiment was performed with the Gram-positive Bacillus thuringiensis AND931 (carrying the conjugative plasmid pXO16), B. thuringiensis 4Q7 (with mobilizable plasmid pC194) and B. thuringiensis GBJ002 (final recipient). A second experiment was carried out with the Gram-negative Escherichia coli CM140 (carrying the conjugative plasmid RP4), E. coli CM1962 (with the mobilizable plasmid pMOL222) and Cupriavidus metallidurans AE815 (final recipient). It was observed by selective platings that plasmid exchange between the Gram-positive bacterial strains occurred in the space flight experiment. It is speculated that the latter plasmid exchange occurs more efficient than in the ground control experiment. No significant differences could be observed between space flight and ground control for the Gram-negative bacteria. The data indicate that plasmid exchange between microorganisms is occurring under space flight conditions. Since microorganisms are endogenous to any spacecraft and their presence considered as a possible jeopardy for manned space exploration, more experiments are needed to evaluate the occurrence and implications of microbial adaptation and evolution via MGE.

Predominance of SHV-5 β-lactamase in enteric bacteria causing community-acquired urinary tract infections in Bosnia and Herzegovina

The beta-lactamases produced by 14 non-duplicate Klebsiella pneumoniae isolates and five Escherichia coli isolates from urine samples obtained from outpatients were characterised by isoelectric focusing, substrate profile determination, PCR and sequencing of bla(SHV) genes. Three E. coli A15 R(+) transconjugants were identified as isolates that produced SHV-5 beta-lactamase. This report is the first description of SHV-5 beta-lactamase among community isolates. Since the isolates showed distinct pulsed-field gel electrophoresis patterns, it was concluded that there was no clonal spread of bla(TEM) and bla(SHV) genes, and that dissemination of the bla(TEM) and bla(SHV) genes was the result of exchange of plasmids among different clones.

Plasmid Diversity in Neisseriae

Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

Holding Back the Tide of Antibiotic Resistance

Another novel approach is being pursued by Wisconsin-based ConjuGon (http://www.conjugon.com/). This eight-person startup is using bacterial sexual reproduction to spread lethal genes in pathogen communities. When the lethal genes are expressed, the bacterial population dies out.“I'm a plasmid biologist,” says Marcin Filutowicz, professor of bacteriology at the University of Wisconsin-Madison, and one of ConjuGon's founders. “Bacterial plasmids are extra chromosomal elements that bacteria don't need to live. They transfer DNA molecules among bacteria. They carry genes which frequently encode for antibiotic resistance. Most of the DNA that is transferred between bacteria is done through plasmid exchange.” ConjuGon engineers specific plasmids with DNA that encodes toxins as well as benign bacteria that are immune to the toxins encoded by the plasmids. When immune bacteria transfer the plasmid that encodes for the toxins to the recipient bacteria, such as a pathogen like Pseudomonas aeruginosa, during conjugation the toxin is expressed, killing the pathogen.“It is a Trojan-horse-like approach where we introduce a DNA molecule from donor to recipient and then express in the recipients multiple toxins or single toxins,” says Filutowicz. “When we express multiple toxins, we reduce the probability for the recipient bacteria to acquire immunity.”ConjuGon is targeting genes that encode for antibiotics that block a certain RNA polymerase at several different locations in bacteria. The company has a patent on “Sigma Binding of RNA Polymerase and Uses Therof” for its method of high-throughput screening for small-molecule drugs that interfere with the binding of transcription factor sigma to the core RNA polymerase. ConjuGon chose this target because it is unlikely that bacteria would mutate in enough multiple ways to evade a compound that interferes with this interaction.Conjugon recently received a phase I SBIR grant from the Army to do research on a treatment for antibiotic-resistant Acineobacter baumannii infections contracted by wounded soldiers in Iraq and Afghanistan. Additionally, Ravi Shankar, associate professor, Department of Surgery and Cell Biology, Neurobiology & Anatomy Burn and Shock Trauma Unit at Loyola University Medical Center, is conducting the proof-of-concept studies on ConjuGon's approach on mice. Shankar is interested in ConjuGon's approach as a specialized or adjunct therapy for patients with burn or trauma wounds who have a long healing process. “Much of the time, especially with burn-wound cases, it is not about life or death. It is about achieving adequate wound coverage with grafts or synthetic material so the person can heal faster,” Shankar says. “But unfortunately, if you have an infection, many of the grafts fail. They lift off.” Conventional antibiotic therapy can be problematic as the antibiotic has to be delivered to the actual wound area of the patient. Thus, a systemic antibiotic is not enough. A majority of time, wound care is delivered via topical antibiotics and antimicrobials, such as topical silver emulsions.Shankar says that in the mouse studies that pathogens were totally eliminated within 30 min of administering ConjuGon treatment. “It actually works…. We have to see if it works on human beings,” Shankar says.

Self-engineering capabilities of bacteria

Under natural growth conditions, bacteria can utilize intricate communication capabilities (e.g. quorum-sensing, chemotactic signalling and plasmid exchange) to cooperatively form (self-organize) complex colonies with elevated adaptability-the colonial pattern is collectively engineered according to the encountered environmental conditions. Bacteria do not genetically store all the information required for creating all possible patterns. Instead, additional information is cooperatively generated as required for the colonial self-organization to proceed. We describe how complex colonial forms (patterns) emerge through the communication-based singular interplay between individual bacteria and the colony. Each bacterium is, by itself, a biotic autonomous system with its own internal cellular informatics capabilities (storage, processing and assessment of information). These afford the cell plasticity to select its response to biochemical messages it receives, including self-alteration and the broadcasting of messages to initiate alterations in other bacteria. Hence, new features can collectively emerge during self-organization from the intracellular level to the whole colony. The cells thus assume newly co-generated traits and abilities that are not explicitly stored in the genetic information of the individuals.

Insights into the genetic organization of the Bacillus mycoides cryptic plasmids pDx14.2 and pSin9.7 deduced from their complete nucleotide sequence

Bacillus mycoides, a member of the Bacillus cereus group of bacteria, can be easily distinguished from close species because of colony shape, made by filaments of cells, resembling fungal hyphae, curving clock- or counterclockwise depending on the strain. Two plasmids, one from a strain curving to the right (pDx14.2), the other from a strain curving to the left (pSin9.7), were sequenced and analyzed for gene content and replication mode. Rolling-circle replication modules and mobilization proteins were found, very similar to those of other plasmids of the B. cereus group bacilli, mostly Bacillus thuringiensis living in the same ecosystem, suggesting active plasmid exchange in nature.

Workshop 3 Role of microbiology (ecological shifts, agent resistance and efficacy)

Workshop 3 Role of microbiology (ecological shifts, agent resistance and efficacy)

Bacterial linguistic communication and social intelligence

Bacteria have developed intricate communication capabilities (e.g. quorum-sensing, chemotactic signaling and plasmid exchange) to cooperatively self-organize into highly structured colonies with elevated environmental adaptability. We propose that bacteria use their intracellular flexibility, involving signal transduction networks and genomic plasticity, to collectively maintain linguistic communication: self and shared interpretations of chemical cues, exchange of chemical messages (semantic) and dialogues (pragmatic). Meaning-based communication permits colonial identity, intentional behavior (e.g. pheromone-based courtship for mating), purposeful alteration of colony structure (e.g. formation of fruiting bodies), decision-making (e.g. to sporulate) and the recognition and identification of other colonies – features we might begin to associate with a bacterial social intelligence. Such a social intelligence, should it exist, would require going beyond communication to encompass unknown additional intracellular processes to generate inheritable colonial memory and commonly shared genomic context.

Microbial Biofilms in the Gut: Visualization by Electron Microscopy and by Acridine Orange Staining

The expression of colonization factors by gut bacteria, the growth rate of gut bacteria, and the rate of plasmid exchange by gut bacteria indicate that biofilms are a normal component of bacterial growth in the large bowel. Further, in vitro experiments demonstrate that growth of normal enteric bacteria in biofilms can be facilitated by secretory IgA (SIgA) and by mucins, 2 major components of the gut milieu. However, biofilms have not been previously observed in the normal gut. In this study, bacterial colonies characteristic of biofilms were observed by electron microscopy in normal rat, baboon, and human gut by electron microscopy. Confirming these results, acridine orange staining of flash-frozen tissues revealed biofilms in the mucus lining along normal gut epithelium. Immunofluorescenct microscopy supported this finding and demonstrated an association between IgA and the biofilms. These findings provide direct evidence that biofilms are present and may play an important role in the commensal relationship between enteric bacteria and their hosts. Hematoxylin and eosin staining of formalin-fixed tissues resulted in dissociation of the luminal contents from the epithelium, suggesting that the association between biofilms and the gut epithelium is sensitive to some conditions used to preserve tissue for histologic evaluation.

Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans -Dominant Mutants

In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids. Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts. These reductions in enzymatic activity usually correlated with increased resistance to the antituberculosis drug isoniazid (INH). However, for the N138S trans-dominant mutant, the catalase-peroxidase activity was significantly decreased while the sensitivity to INH was retained. trans-dominance required katG expression from multicopy plasmids and could not be demonstrated with katG mutants integrated elsewhere on the wild-type M. bovis BCG chromosome. Reversal of the mutant phenotype through plasmid exchange suggested the catalase-peroxidase deficiency occurred at the protein level and that INH resistance was not due to a second site mutation(s). Electrophoretic analysis of KatG proteins from the trans-dominant mutants showed a reduction in KatG dimers compared to WT and formation of heterodimers with reduced activity. The mutants responsible for these defects cluster around proposed active site residues: N138S, T275P, S315T, and D381G. In an attempt to identify mutants that might delimit the region(s) of KatG involved in subunit interactions, C-terminal truncations were constructed (with and without the D381G dominant-negative mutation). None of the C-terminal deletions were able to complement a DeltakatG strain, nor could they cause a dominant-negative effect on the WT. Taken together, these results suggest an intricate association between the amino- and carboxy-terminal regions of KatG and may be consistent with a domain-swapping mechanism for KatG dimer formation.

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production.

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.

Evolution of multiresistance in nontyphoid salmonella serovars from 1984 to 1998 in Argentina.

Molecular evolution of multiresistance in nontyphoid Salmonella spp. was investigated with 155 isolates obtained in Argentina from 1984 to 1998. In 74 isolates obtained from 1984 to 1988 resistance was associated with the presence of Tn3, Tn9, class I (In0) and II (Tn7) integrons, and the aac(3)-IIa gene. Extended-spectrum cephalosporin (ESC) resistance in Salmonella spp. emerged in 1989, and 81 isolates resistant to at least one ESC and one aminoglycoside were collected thereafter. Among these, two patterns of antimicrobial resistance mechanisms were found: from 1989 to 1992, resistance was related to the spreading of Tn1331 and bla(CTX-M-2), in addition to the persistence of In0 and Tn7. From 1993 to 1998, several integrons were added to the first pattern and three integron groups (IG), namely, IG1 (38% of the isolates), IG2 (51%), and IG3 (11%), were identified. At least two beta-lactamase genes were detected in 65% of the isolates (after 1989) by PCR analysis. Furthermore, five beta-lactamase genes, bla(CTX-M-(2)), bla(OXA-9), bla(OXA-2), bla(TEM-1), and bla(PER-2), were found in two isolates. The bla(CTX-M-2) gene was found in several complex sulI-type integrons with different rearrays within the variable region of class I integrons, suggesting evolution of these integrons in nontyphoid Salmonella. In conclusion, progressive acquisition and accumulation of plasmid-mediated resistance determinants occurred from 1984 to 1998 in nontyphoid Salmonella isolates of the most prevalent serovars from Argentina. It is suggested that antimicrobial resistance mechanisms in these bacteria may have been the consequence of plasmid exchange between Salmonella enterica serovar Typhimurium and Escherichia coli or Shigella flexneri and/or spreading of mobile elements from the nosocomial environment.

Acquisition of Antibiotic Resistance Plasmids by Enterohemorrhagic Escherichia coli O157:H7 within Rumen Fluid

The emergence of antibiotic resistance among important foodborne pathogens like Escherichia coli O157:H7 has become an important issue with regard to food safety. In contrast to the case for Salmonella, antibiotic resistance has been slow in its development in E. coli O157:H7 despite the presence of mobile antibiotic resistance genes in other E. coli organisms that inhabit the same animal host. We set out to determine if rumen fluid influences the transfer of plasmid-mediated, antibiotic resistance to E. coli O157:H7. A commensal E. coli strain from a dairy cow was transformed with conjugative R plasmids and served as the donor in matings with naladixic acid–resistant E. coli O157:H7. R plasmids were transferred from the donor E. coli strain to E. coli O157:H7 in both Luria-Bertani (LB) broth and rumen fluid. R plasmids were transferred at a higher frequency to E. coli O157:H7 during 6 h of incubation in rumen fluid at rates comparable to those in LB broth, indicating that conditions in rumen fluid favor the transfer of the plasmids to E. coli O157. This finding suggests that the cow's rumen is a favorable environment for the genetic exchange of plasmids between microflora and resident E. coli O157:H7 in the bovine host.

Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola.

Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of L-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism’s pathogenesis.

Genetic Characterization of Plasmids Containing Genes Encoding Enzymes of Leucine Biosynthesis in Endosymbionts (Buchnera) of Aphids

The prokaryotic endosymbionts (Buchnera) of aphids are known to provision their hosts with amino acids that are limiting in the aphid diet. Buchnera from the aphids Schizaphis graminum and Diuraphis noxia have plasmids containing leuABCD, genes that encode enzymes of the leucine biosynthetic pathway, as well as genes encoding proteins probably involved in plasmid replication (repA1 and repA2) and an open reading frame (ORF1) of unknown function. The newly reported plasmids closely resemble a plasmid previously described in Buchnera of the aphid Rhopalosiphum padi [Bracho AM, Martínez-Torres D, Moya A, Latorre A (1995) J Mol Evol 41:67-73]. Nucleotide sequence comparisons indicate conserved regions which may correspond to an origin of replication and two promoters, as well as inverted repeats, one of which resembles a rho-independent terminator. Phylogenetic analyses based on amino acid sequences of leu gene products and ORF1 resulted in trees identical to those obtained from endosymbiont chromosomal genes and the plasmid-borne trpEG. These results are consistent with a single evolutionary origin of the leuABCD-containing plasmid in a common ancestor of Aphididae and the lack of plasmid exchange between endosymbionts of different aphid species. Trees for ORF1 and repA (based on both nucleotides and amino acids) are used to examine the basis for leu plasmid differences between Buchnera of Thelaxes suberi and Aphididae. The most plausible explanation is that a single transfer of the leu genes to an ancestral replicon was followed by rearrangements. The related replicon in Buchnera of Pemphigidae, which lacks leuABCD, appears to represent the ancestral condition, implying that the plasmid location of the leu genes arose after the Pemphigidae diverged from other aphid families. This conclusion parallels previously published observations for the unrelated trpEG plasmid, which is present in Aphididae and absent in Pemphigidae. Recruitment of amino acid biosynthetic genes to plasmids has been ongoing in Buchnera lineages after the infection of aphid hosts.

Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedl gene (ssI0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (sIr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (sIr0150, negative control) or glucose availability (sII1382). fedl was found to be critical to Synechocystis PCC6803 viability in spite of sIr0150, sII1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedl could be deleted from all chromosome copies in cells propagating a fedl gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl mutant strains generated after plasmid exchange showed that the C18-C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the Fedl carboxy terminus is important for effective PSI/FedI interactions. The plasmid-shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.