Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis

Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.     

Expression of a Lactose Transposon (Tn951) in Zymomonas mobilis

The potential utility of Zymomonas mobilis as an organism for the commercial production of ethanol would be greatly enhanced by the addition of foreign genes which expand its range of fermentable substrates. We tested various plasmids and mobilizing factors for their ability to act as vectors and introduce foreign genes into Z. mobilis CP4. Plasmid pGC91.14, a derivative of RP1, was found to be transferred from Escherichia coli to Z. mobilis at a higher frequency than previously reported for any other plasmids. Both tetracycline resistance and the lactose operon from this plasmid were expressed in Z. mobilis CP4. Plasmid pGC91.14 was stably maintained in Z. mobilis at 30°C but rapidly lost at 37°C.     

Fermentation of lactose by Zymomonas mobilis carrying a Lac+ recombinant plasmid

Lac⁺ recombinant plasmids encoding a β-galactosidase fused protein and lactose permease of Escherichia coli were introduced Zymomonas mobilis. The fused protein was expressed with 450 to 5,860 Miller units of β-galactosidase activity, and functioned as lactase. Raffinose uptake by Z. mobilis CP4 was enhanced in the plasmid-carrying strain over the plasmid-free strain, suggesting that the lactose permease was functioning in the organism. Z. mobilis carrying the plasmid could produce ethanol from lactose and whey, but could not grow on lactose as the sole carbon source. It was found that the growth of the organism was inhibited by either galactose of the galactose liberated from lactose.     

Construction of a shuttle vector for Zymomonas mobilis

Summary An Escherichia coli-Zymomonas mobilis shuttle vector was constructed from a 15.5 kb native plasmid of ZM6 00 and the E. coli plasmid, pBR329. Integrative transfer of this shuttle vector from E. coli to Z. mobilis was achieved with the aid of the mobilizing plasmid, pRK2013. The shuttle vector was stable in Z. mobilis for at least 300 generations without antibiotic selection.Offprint requests to: S. F. Delaney     

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.     

Cloning and expression inEscherichia coli of arecA-like gene fromZymomonas mobilis

Intergeneric complementation ofEscherichia coli recA mutants was used to identify recombinant plasmids, within a genomic library derived fromZymomonas mobilis, that carryZ. mobilis recA-like gene. Screening of 1100 individualE. coli strains revealed four clones expressing therecA⁺ character. On restriction analysis, all four recombinant plasmids were found to be related and to exhibit a common 6.7-kb fragment. Consequently, one of the four recombinant plasmids, pZR27, was selected for further characterization. When introduced intoE. coli recA mutants, pZR27 restored resistance to methyl methane sulfonate, mitomycin-C, and UV irradiation, as well as recombination proficiency when measured by standard Hfr-mediated conjugation. The clonedrecA-like gene also restored the spontaneous and mitomycin-C-induced phage production. The origin of the insert in pZR27 from the chromosome ofZ. mobilis was confirmed by Southern transfer and DNA hybridization. However, no homology was found between therecA ofE. coli andZ. mobilis chromosomal insert DNA. TheZ. mobilis recA-like gene also encoded a major polypeptide of 38-kDa on SDS-PAGE.     

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Summary Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of -galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed -galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.     

Complete Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Centrotype ATCC 29191

Zymomonas mobilis is an ethanologenic bacterium that has been studied for use in biofuel production. Of the sequenced Zymomonas strains, ATCC 29191 has been described as the phenotypic centrotype of Zymomonas mobilis subsp. mobilis, the taxon that harbors the highest ethanol-producing Z. mobilis strains. ATCC 29191 was isolated in Kinshasa, Congo, from palm wine fermentations. This strain is reported to be a robust levan producer, while in recent years it has been employed in studies addressing Z. mobilis respiration. Here we announce the finishing and annotation of the ATCC 29191 genome, which comprises one chromosome and three plasmids.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis

A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201–3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.     

Genetics and genetic engineering ofZymomonas mobilis

Present knowledge on the genetics of the ethanologenic anaerobeZymomonas mobilis includes background information on: size, restriction, and to some extent hybridization, analysis of indigenous plasmids; mutagenesis and isolation of a wide variety of auxotrophic, drug resistant and conditional mutants; construction of shuttle cloning vectors able to replicate and express inZ. mobilis; development of gene transfer systems based on conjugal mobilization of plasmids fromEscherichia coli donors toZ. mobilis; expression of heterologous genes inZ. mobilis; cloning and analysis of genes encoding enzymes of the Entner-Doudoroff pathway. Moreover, preliminary data on recombinational repair mechanisms and plasmid stability, which are now available, makeZ. mobilis an attractive model system for molecular genetic research and, furthermore, they contribute towards expansion of the substrate and product range of this industrial microorganism.G.A. Sprenger is with the Institut für Biotechnologie I, Forschungszentrum, KFA Julich, Postfach 1913, D-5170 Julich, Germany. M.A. Typas is with the Department of Biochemistry, Molecular & Cellular Biology and Genetics. University of Athens, Kouponia 105 71 Athens, Greece. C. Drainas is with the Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of loannina, 451 10 loannina, Greece.     

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration,and Acquisition of the Plasmid Results in a Variable Cost of Fitness

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.     

Recruiting alternative glucose utilization pathways for improving succinate production

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS^? strains, and their impacts on glucose utilization and succinate production were compared. All PTS^? strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS^? strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk _{E. coli} (2.13 g/L?h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk _{E. coli} . On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L?h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.     

Transformation of Zymomonas mobilis by a hybrid plasmid

The transformation of Zymomonas mobilis by plasmid DNA was achieved using a modification of the CaCl₂ method for Escherichia coli. The highest frequency of transformation obtained was 5 × 10³ transformants/μg DNA. The success of the method depended upon the use of a plasmid which is a cointegrate between a Z. mobilis cryptic plasmid and an E. coli plasmid carrying two selectable drug resistance markers.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics     

Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

The Zymomonas mobilis ZM4 strain with excellent ethanol‐producing capabilities was the first strain of Z. mobilis, which was sequenced. This strain is resistant to transformation, and no previous study has shown a detailed protocol for electrotransfer of ZM4 with foreign DNA. In this work, many electrical and biological parameters were selected and evaluated in order to optimize the electrotransformation of ZM4. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants/μg DNA were separately achieved with shuttle plasmid pZB21‐mini (3082 bp), pZB21 (5930 bp), pZA22 (6994 bp) and broad‐host‐range vector pBBR1MCS‐2 (5144 bp) all prepared from Escherichia coli JM110. The crucial factors affecting the transformation efficiency included the source of the plasmid (the best strain was ZM4), origin and size of the plasmids, growth phase of the cells (the most ideal phase was early log phase with OD₆₀₀ of 0.3–0.4), the electric field strength (generally 11.75 kV/cm–13.25 kV/cm) and the recovery time (3–24 h). Further, based upon the optimal transformation protocol mentioned above for replicative plasmids in ZM4, (i) the electrotransformation by recombinant plasmid pBBR1MCS‐2‐Pgap‐FLP (6880 bp) was an immediate success with the transformation efficiency 10² transformants/μg DNA; (ii) the site‐specific integration efficiencies (expressed in terms of “per μg of DNA”) of 3–6 integrating transformants was obtained using the integrating plasmid pBR328‐ldhR‐cml‐ldhL (7447 bp). This study will assist genetic and biotechnological research of ZM4 and other Z. mobilis strains by providing information about suitable vectors and a more universal and reliable procedure for introducing DNA into this strain.     

Plasmid size can affect the ability of Escherichia coli to produce high-quality plasmids

Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8?kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.