Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli

The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.     

Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7.

The translation inhibitor microcin C7 (MccC7) is a linear heptapeptide whose N terminus has been replaced by an N-formyl group and whose C terminus has been replaced by the phosphodiester of 5'-adenylic acid and n-aminopropanol (J. I. Guijarro, J. E. González-Pastor, F. Baleux, J. L. San Millán, M. A. Castilla, M. Rico, F. Moreno, and M. Delepierre, J. Biol. Chem. 270:23520-23532, 1995). MccC7 production and immunity determinants lie on a 6.2-kb region of the Escherichia coli plasmid pMccC7. This region was entirely sequenced. It contains six open reading frames, which were shown to be true genes by different complementary approaches. Five genes, mccABCDE, which are transcribed in the same direction, are required to produce mature extracellular microcin. The sixth gene, mccF, adjacent to mccE, is transcribed in the opposite direction and encodes specific self-immunity. Genes mccA to -E constitute an operon transcribed from a promoter (mccp) located upstream of mccA. mccA is 21 nucleotides long and encodes the unmodified heptapeptide (J. E. González-Pastor, J. L. San Millán, and F. Moreno, Nature [London] 369:281, 1994). A comparison of predicted gene polypeptide products with those included in databases shows that an 81-amino-acid stretch of MccB is strikingly homologous to fragments of the same length of proteins ThiF and ChlN from E. coli, HesA from Anabaena sp. strain PCC7120, and UBA1, the ubiquitin-activating enzyme from different eukaryotic species. MccC displays several hydrophobic domains, suggesting a transmembrane location. The carboxyl end of MccE displays 41.2% identity with RimL, a protein required to acetylate the ribosome protein L12 from E. coli. In the absence of the other mcc genes, mccA impairs the growth of host cells, suggesting that unmodified MccA has antibiotic activity. A model for MccC7 biosynthesis, export, and immunity is proposed.     

ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification.

Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene. A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype. The plasmid led to overproduction of OmpT protein and protease activity in the E. coli K-12 and B strains we used, but only weak expression in the E. coli C strain, C1757. This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis. A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes. Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine. Such strains may be useful for purification of other proteins expressed in E. coli.     

Green fluorescent protein labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for safety-related studies

Many food safety-related studies require tracking of introduced foodborne pathogens to monitor their fate in complex environments. The green fluorescent protein (GFP) gene (gfp) provides an easily detectable phenotype so has been used to label many microorganisms for ecological studies. The objectives of this study were to label major foodborne pathogens and related bacteria, including Listeria monocytogenes, Listeria innocua, Salmonella, and Escherichia coli O157:H7 strains, with GFP and characterize the labeled strains for stability of the GFP plasmid and the plasmid's effect on bacterial growth. GFP plasmids were introduced into these strains by a CaCl(2) procedure, conjugation or electroporation. Stability of the label was determined through sequential propagation of labeled strains in the absence of selective pressure, and rates of plasmid-loss were calculated. Stability of the GFP plasmid varied among the labeled species and strains, with the most stable GFP label observed in E. coli O157:H7. When grown in nonselective media for two consecutive subcultures (ca. 20 generations), the rates of plasmid loss among labeled E. coli O157:H7, Salmonella and Listeria strains ranged from 0%-30%, 15.8%-99.9% and 8.1%-93.4%, respectively. Complete loss (>99.99%) of the plasmid occurred in some labeled strains after five consecutive subcultures in the absence of selective pressure, whereas it remained stable in others. The GFP plasmid had an insignificant effect on growth of most labeled strains. E. coli O157:H7, Salmonella and Listeria strains can be effectively labeled with the GFP plasmid which can be stable in some isolates for many generations without adversely affecting growth rates.     

Study of the regulation of crp gene expression in Escherichia coli K12

The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.     

Increased production of the protein antigen of Brucella melitensis in Escherichia coli K-12 cells

The production of Brucella melitensis protein antigen with molecular weight of 38 kD in Escherichia coli K-12 cell lysates has been studied by immunoblotting with various antisera. E. coli strains differed by the vector plasmid and the size of B. melitensis 565 DNA fragment with 38 kD protein gene, cloned in this plasmid. The immunoblotting analysis detected increased production of 38 kD protein in the recombinant GSE830 strain in comparison with the B. melitensis strain 565, from which the gene was cloned, and other E. coli strains containing this protein gene. The production of 38 kD protein was determined by the size of the cloned B. melitensis 565 DNA fragment with this protein gene, but not by the conditions of culturing.     

Relationship between O-serogroup and presence of pathogenic factor genes in Escherichia coli

A total of 383 isolates of serogroup-based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes. The serogroup-based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers. The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%). The difference was not significant. The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA-positive EPEC strains from patients but was not detected in the 9 strains from carriers. In serogroup-based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers. The 4 ipaH-positive strains possessed the invasive plasmid. These results suggested that the serogroup-based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene.     

通过一种新方法使T7基因Ⅰ置换araBAD基因簇

目的：用T7RNA聚合酶基因T7基因Ⅰ置换大肠杆菌基因组中的araBAD基因簇。方法：通过分析大肠杆菌基因组中araBAD基因序列,设计两侧同源臂,构建高拷贝数的打靶质粒,并且在这个质粒上的打靶片段两侧各加上一个归巢内切酶位点;将辅助质粒和打靶质粒同时转化到宿主体内,在L-阿拉伯糖的诱导下表达归巢内切酶和Red重组酶,实现体内重组,使T7基因I置换araBAD基因簇。结果：在4株不同的大肠杆菌中用T7基因I置换了araBAD基因簇。结论：成功地将T7基因Ⅰ整合到araBAD基因位置,为构建PAra-PT7表达系统奠定了基础。     

Characterization of the binding of diarrheagenic strains of E. coli to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts

Diarrheagenic Escherichia coli were able to bind to plant surfaces, including alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana seedlings incubated in water. The characteristics of the binding differed with the bacterial strain examined. Laboratory K12 strains of E. coli failed to show significant binding to any of the plant surfaces examined, suggesting that some of the genes present and expressed in pathogenic strains and absent or unexpressed in K12 strains may be required for binding to plants. When a plasmid carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid carrying the operons that encode the synthesis of curli (csgA-G) was introduced into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction in their ability to bind to sprouts. Thus, the production of curli appears to be sufficient to allow K12 strains to bind, but curli are not necessary for the binding of pathogenic strains, suggesting that pathogenic strains may have more than one mechanism for binding to plant surfaces.     

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.     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

[目的]为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略.[方法]将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒.将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基...     

Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 025.H42

Two enterotoxigenic Escherichia coli strains of serotype 0.25.H42 that produced coli surface associated antigens CS4 and CS6 hybridized with a probe containing the cfaD sequence that regulates expression of colonization factor antigen CFA/I. Transformation of a cloned cfaD gene into some derivatives of the strains that were negative for CS4 and CS6 resulted in expression of CS4 but not CS6. By hybridization the sequence that regulated CS4 production in the wild type 025 strains was located on a plasmid that also encoded the CS6 antigen. The structural genes for the CS4 antigen were on a separate plasmid. The 025 strains carried a third plasmid encoding enterotoxin production which was therefore unlinked to regulation sequences or genes encoding CS antigens.     

Effect of the gene for bacterial hemoglobin vhb on the effectiveness of the process of Escherichia coli-Streptomyces interspecies conjugation and production of antibiotics in streptomycetes

The bacterial hemoglobin vhb gene was cloned from sliding bacterium Vitreoscilla sp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhb gene was transferred from Escherichia coli to some Streptomyces strains, producers of antibiotics, by the method of intergeneric conjugation using conjugative-integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhb gene on processes of conjugation and antibiotic production in a number of examined strains was shown.     

Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine.

We report that rfe mutants of wild-type strains of Escherichia coli O7, O18, O75, and O111 did not express O-specific polysaccharide unless the rfe mutation was complemented by a cloned rfe gene supplied in a plasmid. The O polysaccharides in these strains are known to have N-acetylglucosamine (GlcNAc) in their O repeats. In addition, in vitro transferase assays with bacterial membranes from either the O7 wild-type strain or its isogenic rfe mutant showed that GlcNAc is the first carbohydrate added onto the lipid acceptor in the assembly of the O7 repeat and that this function is inhibited by tunicamycin. Our results indicate that the rfe gene product is a general requirement for the synthesis of O polysaccharides containing GlcNAc.     

Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells

When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.     

Plasmid-mediated conjugative transfer of Klebsiella sp. rcs genesable to induce colanic acid capsular polysaccharide biosynthesis in Escherichia coli

Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.