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Abstract Background Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks. Results By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme. Conclusions By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.

Abstract Background Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential. Results We analyze an extreme case of toxic plasmid clone instability, and show that random host IS element hopping, causing inactivation of the toxic cloned sequences, followed by automatic selection of the fast-growing mutants, can prevent the maintenance of a clone developed for vaccine production. Analyzing the molecular details, we identify a hydrophobic protein as the toxic byproduct of the clone, and show that IS elements spontaneously landing in the cloned fragment relieve the cell from the stress by blocking transcription of the toxic gene. Bioinformatics analysis of sequence reads from early shotgun genome sequencing projects, where clone libraries were constructed and maintained in E. coli, suggests that such IS-mediated inactivation of ectopic genes inhibiting the growth of the E. coli cloning host might happen more frequently than generally anticipated, leading to genomic instability and selection of altered clones. Conclusions Delayed genetic adaptation of clean-genome, IS-free MDS42 host improves maintenance of unstable genetic constructs, and is suggested to be beneficial in both laboratory and industrial settings.

Artificial simplification of bacterial genomes is thought to have the potential to yield cells with reduced complexity, enhanced genetic stability, and improved cellular economy. Of these goals, economical gains, supposedly due to the elimination of superfluous genetic material, and manifested in elevated growth parameters in selected niches, have not yet been convincingly achieved. This failure might stem from limitations of the targeted genome reduction approach that assumes full knowledge of gene functions and interactions, and allows only a limited number of reduction trajectories to interrogate. To explore the potential fitness benefits of genome reduction, we generated successive random deletions in E. coli by a novel, selection-driven, iterative streamlining process. The approach allows the exploration of multiple streamlining trajectories, and growth periods inherent in the procedure ensure selection of the fittest variants of the population. By generating single- and multiple-deletion strains and reconstructing the deletions in the parental genetic background, we showed that favourable deletions can be obtained and accumulated by the procedure. The most reduced multiple-deletion strain, obtained in five deletion cycles (2.5% genome reduction), outcompeted the wild-type, and showed elevated biomass yield. The spectrum of advantageous deletions, however, affecting only a few genomic regions, appears to be limited.

The ideal bacterial chassis provides a simplified, stable and predictable host environment for synthetic biological circuits. Mutability and evolution can, however, compromise stability, leading to deterioration of artificial genetic constructs. By eliminating certain sources of instability, these undesired genetic changes can be mitigated. Specifically, deletion of prophages and insertion sequences, nonessential constituents of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization. Here, we sought to establish a rapid methodology to improve the stability of microbial hosts. The novel workflow involves genome shuffling between a mobile genetic element-free strain and the target cell, and subsequent rounds of CRISPR/Cas-assisted MAGE on multiplex targets. The power and speed of the procedure was demonstrated on E. coli BL21(DE3), a host routinely used for plasmid-based heterologous protein expression. All 9 prophages and 50 insertion elements were efficiently deleted or inactivated. Together with additional targeted manipulations (e.g., inactivation of error-prone DNA-polymerases), the changes resulted in an improved bacterial host with a hybrid (harboring segments of K-12 DNA), 9%-downsized and clean genome. The combined capacity of phage-mediated generalized transduction and CRISPR/Cas-selected MAGE offers a way for rapid, large scale editing of bacterial genomes.

The release of the first complete microbial genome sequences at the end of the past century opened the way for functional genomics and systems-biology to uncover the genetic basis of various phenotypes. The surge of available sequence data facilitated the development of novel genome editing techniques for system-level analytical studies. Recombineering allowed unprecedented throughput and efficiency in microbial genome editing and the recent discovery and widespread use of RNA-guided endonucleases offered several further perspectives: (i) previously recalcitrant species became editable, (ii) the efficiency of recombineering could be elevated, and as a result (iii) diverse genomic libraries could be generated more effectively. Supporting recombineering by RNA-guided endonucleases has led to success stories in metabolic engineering, but their use for system-level analysis is mostly unexplored. For the full exploitation of opportunities that are offered by the genome editing proficiency, future development of large scale analytical procedures is also vitally needed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of the metabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

Genome engineering strategies--such as genome editing, reduction and shuffling, and de novo genome synthesis--enable the modification of specific genomic locations in a directed and combinatorial manner. These approaches offer an unprecedented opportunity to study central evolutionary issues in which natural genetic variation is limited or biased, which sheds light on the evolutionary forces driving complex and extremely slowly evolving traits; the selective constraints on genome architecture; and the reconstruction of ancestral states of cellular structures and networks.

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large-scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole-genome sequencing of laboratory-evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton-motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF-dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade-offs under antibiotic selection.

Although both genotypes with elevated mutation rate (mutators) and mobilization of insertion sequence (IS) elements have substantial impact on genome diversification, their potential interactions are unknown. Moreover, the evolutionary forces driving gradual accumulation of these elements are unclear: Do these elements spread in an initially transposon-free bacterial genome as they enable rapid adaptive evolution? To address these issues, we inserted an active IS1 element into a reduced Escherichia coli genome devoid of all other mobile DNA. Evolutionary laboratory experiments revealed that IS elements increase mutational supply and occasionally generate variants with especially large phenotypic effects. However, their impact on adaptive evolution is small compared with mismatch repair mutator alleles, and hence, the latter impede the spread of IS-carrying strains. Given their ubiquity in natural populations, such mutator alleles could limit early phase of IS element evolution in a new bacterial host. More generally, our work demonstrates the existence of an evolutionary conflict between mutation-promoting mechanisms.

The last few years have witnessed rapid progress in bacterial genome engineering. The long-established, standard ways of DNA synthesis, modification, transfer into living cells, and incorporation into genomes have given way to more effective, large-scale, robust genome modification protocols. Expansion of these engineering capabilities is due to several factors. Key advances include: (i) progress in oligonucleotide synthesis and in vitro and in vivo assembly methods, (ii) optimization of recombineering techniques, (iii) introduction of parallel, large-scale, combinatorial, and automated genome modification procedures, and (iv) rapid identification of the modifications by barcode-based analysis and sequencing. Combination of the brute force of these techniques with sophisticated bioinformatic design and modeling opens up new avenues for the analysis of gene functions and cellular network interactions, but also in engineering more effective producer strains. This review presents a summary of recent technological advances in bacterial genome engineering., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bacteriophages, the historic model organisms facilitating the initiation of molecular biology, are still important candidates of numerous useful or promising biotechnological applications. Development of generally applicable, simple and rapid techniques for their genetic engineering is therefore a validated goal. In this article, we report the use of bacteriophage recombineering with electroporated DNA (BRED), for the first time in a coliphage. With the help of BRED, we removed a copy of mobile element IS1, shown to be active, from the genome of P1vir, a coliphage frequently used in genome engineering procedures. The engineered, IS-free coliphage, P1virdeltaIS, displayed normal plaque morphology, phage titre, burst size and capacity for generalized transduction. When performing head-to-head competition experiments, P1vir could not outperform P1virdeltaIS, further indicating that the specific copy of IS1 plays no direct role in lytic replication. Overall, P1virdeltaIS provides a genome engineering vehicle free of IS contamination, and BRED is likely to serve as a generally applicable tool for engineering bacteriophage genomes in a wide range of taxa., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Background: Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential., Results: We analyze an extreme case of toxic plasmid clone instability, and show that random host IS element hopping, causing inactivation of the toxic cloned sequences, followed by automatic selection of the fast-growing mutants, can prevent the maintenance of a clone developed for vaccine production. Analyzing the molecular details, we identify a hydrophobic protein as the toxic byproduct of the clone, and show that IS elements spontaneously landing in the cloned fragment relieve the cell from the stress by blocking transcription of the toxic gene. Bioinformatics analysis of sequence reads from early shotgun genome sequencing projects, where clone libraries were constructed and maintained in E. coli, suggests that such IS-mediated inactivation of ectopic genes inhibiting the growth of the E. coli cloning host might happen more frequently than generally anticipated, leading to genomic instability and selection of altered clones., Conclusions: Delayed genetic adaptation of clean-genome, IS-free MDS42 host improves maintenance of unstable genetic constructs, and is suggested to be beneficial in both laboratory and industrial settings.

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

E. coli K-12, being one of the best understood and thoroughly analyzed organisms, is the workhorse of genetic, biochemical, and systems biology research, as well as the platform of choice for numerous biotechnological applications. Genome minimization/remodeling is now a feasible approach to further enhance its beneficial characteristics for practical applications. Two genome engineering techniques, a lambda Red-mediated deletion method and a suicide (conditionally replicative) plasmid-based allele replacement procedure are presented here. These techniques utilize homologous recombination, and allow the rapid introduction of virtually any modifications in the genome.

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

Quantitative assessment of the spontaneous or induced genomic mutation rate, a fundamental evolutionary parameter, usually requires the use of well-characterized mutant selection systems. Although there is a great number of genetic selection schemes available in Escherichia coli, the selection of D-cycloserine resistant mutants is shown here to be particularly useful to yield a general view of mutation rates and spectra. The combination of a well-defined experimental protocol with the Ma-Sandri-Sarkar maximum likelihood method of fluctuation analysis results in reproducible data, adequate for statistical comparisons. The straightforward procedure is based on a simple phenotype-genotype relationship, and detects mutations in the single-copy, chromosomal cycA gene, involved in the uptake of D-cycloserine. In contrast to the widely used rifampicin resistance assay, the procedure selects mutations which are neutral in respect of cell growth. No specific genetic background is needed, and practically the entire mutation spectrum (base substitutions, frameshifts, deletions, insertions) can simultaneously be measured. A systematic analysis of cycA mutations revealed a spontaneous mutation rate of 6.54 x 10(-8) in E. coli K-12 MG1655. The mutation spectrum was dominated by point mutations (base substitutions, frameshifts), spread over the entire gene. IS insertions, caused by IS1, IS2, IS3, IS4, IS5 and IS150, represented 24% of the mutations.

Our goal is to construct an improved Escherichia coli to serve both as a better model organism and as a more useful technological tool for genome science. We developed techniques for precise genomic surgery and applied them to deleting the largest K-islands of E. coli, identified by comparative genomics as recent horizontal acquisitions to the genome. They are loaded with cryptic prophages, transposons, damaged genes, and genes of unknown function. Our method leaves no scars or markers behind and can be applied sequentially. Twelve K-islands were successfully deleted, resulting in an 8.1% reduced genome size, a 9.3% reduction of gene count, and elimination of 24 of the 44 transposable elements of E. coli. These are particularly detrimental because they can mutagenize the genome or transpose into clones being propagated for sequencing, as happened in 18 places of the draft human genome sequence. We found no change in the growth rate on minimal medium, confirming the nonessential nature of these islands. This demonstration of feasibility opens the way for constructing a maximally reduced strain, which will provide a clean background for functional genomics studies, a more efficient background for use in biotechnology applications, and a unique tool for studies of genome stability and evolution.

Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5alpha background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Deltafur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.

The SinI and EcoRII DNA methyltransferases recognize sequences (GG(A)/(T)CC and CC(A)/(T)GG, respectively), which are characterized by an (A)/(T) ambiguity. Recognition of the A.T and T.A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine.C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G.C or a C.G base pair in the center of the recognition sequence (GG(G)/(C)CC and CC(G)/(C)GG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG(A)/(T)CC and GG(G)/(C)CC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that (A)/(T) versus (G)/(C) discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2microm plasmid-derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100kb apart. Moreover, 2microm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2microm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

The effectiveness of seromyotomy of the lesser curvature of the stomach--the simplified version of proximal selective vagotomy--was investigated in eleven dogs. Decreased acid secretion was proved with congo red test and pH measuring by glass electrode. No significant damage to the motor function of the stomach was found with scintigraphy. Histological examinations revealed neurofibre degeneration peripherally to the seromyotomy line after peripheral vagotomy and vacuolar degeneration in the ganglion cells and amputation neuromas in the seromyotomy line.

Objective: To report our countrywide experience of laparoscopic cholecystectomy with particular reference to complications., Design: National multicentre retrospective study., Setting: 71 university departments and public hospitals in Hungary., Subjects and Interventions: 13,833 patients operated on between 1 January 1990 and 31 December 1993. Follow up by questionnaire., Results: 732 patients (5.3%) required conversion to open cholecystectomy, urgently because of intraoperative complications in 204 (1.5%), electively for acute or chronic inflammation or obscure anatomy in 441 (3.2%), for unexpected findings in 72 (0.5%) and for technical problems in 15 (0.1%). There were postoperative complications in 598 (4.3%) but reoperation was indicated in only 154 patients (1.1%). There were bile duct injury in 81 (0.6%) and 199 bleeds (1.4%) which required conversion in 102 patients (0.7%) and reoperation in 38 (0.3%). 36 of the 181 postoperative recognized bile leaks required reoperation (20%). 19 patients died (0.1%)., Conclusions: The morbidity and the mortality of laparoscopic cholecystectomy are better than after the open operation. The 2-6 times higher risk of bile duct injury mentioned in early studies was not confirmed.

As enthusiasm for laparoscopic surgery has grown, laparoscopic approaches to the groin hernia have evolved. The most widely accepted laparoscopic repair employs the placement of a large sheet of mesh in a preperitoneal position to cover potential hernia spaces. Between March 1994 and February 1997 160 inguinal and 3 femoral hernia were operated of an transabdominal preperitoneal (TAPP) polipropylen mesh. 131 patients were operated (128 males and 3 females, ranging in age from 19 to 82 years), 31 (23%) of them had bilateral hernias. Recurrent hernia was the indication in 52 (32%) cases. Average operating time for unilateral repair was 80 minutes and for bilateral repairs was 108 minutes. Postoperative complications included 7 (4.3%) cases of transient neuralgias, 20 (12%) cord/scrotal transient seromas-hematomas and 2 (1.2%) hydrocele. The 5 (3.1%) early recurrences were considered to be caused by technical inexperience and/or too small prosthetic patch. The laparoscopic hernioplasty has definitive advantage: minimal postoperative pain, short hospital stay (average postoperative time of hospitalization 3.1 days) and early restoration of full physical activity (in 1 to 2 weeks). The method should be considered as a potential "best option" in patients with recurrences and bilateral inguinofemoral hernias.

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

The development of minimally invasive surgery brought up the challenge: to repair the frequent inguino-femoral hernias laparoscopically. The authors performed 65 laparoscopic hernioplasties in one year: "transabdominal preperitoneal" technique was used in 61 cases und "intraperitoneal onlay mesh" in 4 cases. Fifty-three patients were operated on, 12 of them had bilateral hernias. Recurrent hernia was the indication in 22 patients (34%). The average operating time was 102 and 144 minutes in the unilateral and the bilateral cases, respectively. There was no wound infection, or general complication. Spontaneously dissolving seroma/hematoma of the spermatic cord was noticed and detected by ultrasound in 5 patients (7.7%). The neuralgia caused by the irritation of the nerves of the region in 4 patients (6.1%) disappeared without sequels after treatment with vitamins B. The 2 early recurrences (3.2%), considered to be caused by technical inexperience, these patients were treated successfully with the "intraperitoneal onlay mesh" technique. In the authors' opinion there are definite advantages of laparoscopic hernioplasty, namely the minimal postoperative pain, early mobilization, shorter hospital stay and early restoration of full physical activity (in 1-2 weeks) as well as the known disadvantages of this technique (narcosis, longer operative time, intraperitoneal procedure, higher costs).

There is no disease that would have as many and variable complications as Crohn's disease. One of the most common complications is bowel obstruction which can be caused by the angulation of the bowel or by inflammation, or by formation of granulation tissue (32.3%). Very common is the formation of fistulae amongst the bowels involved and other abdominal organs, and also entero-cutaneous fistulae occur frequently (11.3-14.4%). The frequency of complications is increasing with the duration of the illness. If they are life-threatening, only surgical treatment can help. Surgical treatment is also indicated when conservative treatment fails. The most commonly used surgical interventions are bowel resection and, recently, the plasty of stenotic areas. The operative mortality (3.7%) is influenced by the length of the disease and by the numbers of complications. Recurrence is very common in Crohn's disease (30.1-34.9%). The mortality rate of the second operation is 17.5%. The prognosis is usually poor because recurrence can occur years after the primary operation. In Hungary, the frequency of surgically treated patients with Crohn's disease is low, they count for 0.06% of all general surgical operations.

The development of minimally invasive surgery has accepted the challenge by having tried to repair inguino-femoral hernias laparoscopically. The authors performed 65 laparoscopic hernioplasties in one year. "Transabdominal preperitoneal" technique was applied in 61 cases and "intraperitoneal onlay mesh" in 4 cases. Fifty-three patients were operated, 12 of them had bilateral hernias. Recurrent hernia was the indication in 22 patients (34%). The average operating time was 102 and 144 minutes in the unilateral and the bilateral cases, respectively. There was neither wound infection, nor general complication. Spontaneously dissolving seroma/haematoma of the spermatic cord was noticed (detected by ultrasound) in 5 patients (7.7%). The neuralgia caused by the irritation of the nerves of the region in 4 patients (6.1%) disappeared without sequels after treatment with vitamins B. The 2 early recurrences (3.2%) were considered to be caused by technical unexperience; the affected patients were treated successfully with the "intraperitoneal onlay mesh" technique. It is emphasized that laparoscopic hernioplasty has definite advantages, namely minimal postoperative pain, early mobilization, short hospital stay and early restoration of full physical activity (in 1 to 2 weeks). On the other hand, general anaesthesia and intraperitoneal invasion are required and the operation consumes much time and cost.

The Swedish surgeon Jacobaeus was the first to use a lighted cystoscope for the lysis of pleural adhesions /5/. The same author reported, in 1921, five cases of intrathoracic malignancies diagnosed by thoracoscopy /6/. After the appearance of video-assisted cholecystectomy and abdominal surgery, video-assisted thoracic surgery (VATS) also occupied a major share in thoracic surgery. It was long ago that we introduced a thoracoscope (not the video-assisted type) for thoracic intervention. In 1988 we published 24 cases of thoracic sympathectomies in which we used the thoracoscope /16/.

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

The authors investigated the efficiency of the seromyotomy of the lesser curvature of the stomach in 11 dogs. Reduction of the acid production was verified by glass electrode pH measuring of the gastric mucosa. Scintigraphic examination showed that the operation did not damage the motor function (motility) of the stomach. The fact of peripheral vagotomy was established by histological examination: degeneration of nerve fibres was detected on the peripheral side of the line of the seromyotomy, vacuolar degeneration was found in the cells of parasympathetic ganglions, and amputation neurinomas were shown in the line of the seromyotomy.

Traditional cholecystectomy has been the standard surgical treatment of the gallstone disease for more than 100 years. The technical development led to a new surgical procedure and its rapid acceptance. This is laparoscopic cholecystectomy. Its application is becoming widespread in therapy too. But most of the surgeons are lack of technical experiences in this field. Currently it restricts the indications those are anyway the same of standard cholecystectomy. Besides its many advantages, laparoscopic cholecystectomy has its own disadvantages and being an invasive procedure, there are possibilities of complications. The latest can be reduced by the adequate choice of patients, the careful learning of the operative technic and by turning to open surgery (conversion) when it is necessary. Its morbidity is nearly equal to complications of standard cholecystectomy, but mortality rate is lower (0.05-0.2%). Our morbidity of performed 300 laparoscopic cholecystectomies was 6.4%. We had no death. The hospitalization became as short as 4 days. Our early clinical results (90%) are the same of traditional cholecystectomy. Laparoscopic cholecystectomy as a new surgical procedure involves the efficiency of the standard cholecystectomy and the noninvasive endoscopic technic. Laparoscopic cholecystectomy performed by well trained surgeons is a safe surgical procedure, its early results are excellent and makes the choice of surgical treatment, used in bile surgery richer.

Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.

A vector was designed to allow predetermined and precise fusion between two cloned genes by constructing a cassette with two unique class-IIS restriction sites, 5'-ACCTGC-3' (BspMI) and 5'-CCGGATG-3' (FokI overlapping with MspI), arranged back-to-back in a divergent manner and inserted at the HincII site of a multiple cloning site (MCS) in plasmid pUC18 or analogous vehicle. Two DNA fragments or genes to be precisely fused are cloned into the MCS parts located on each side of the cassette containing the two unique class-IIS restriction sites. The BspMI and MspI/FokI sites are used to generate unidirectional deletions of the genes as previously described [Hasan et al., Gene 50 (1986) 55-62; Pósfai and Szybalski, Nucleic Acids Res. 16 (1988) 6245]. The precisely trimmed genes are ligated after the cassette containing the unique class-IIS restriction sites are excised with BspMI + FokI and the termini were blunted with mung-bean nuclease. This method was used to construct a hybrid methyltransferase (MTase) from the M.BspRI and M.BsuRI MTases, which share a high degree of overall homology (about 65%) and have the identical sequence specificity (5'-GGmCC-3'). A hybrid MTase composed of the N-terminal part of M.BspRI and the C-terminal part of M.BsuRI was constructed and found to be fully functional.

Analyzing the patient material of 506 patients with gastric resection, authors deal with the early complication of resection, its diagnosis and treatment. They review the complications, their frequency and mortality in their own material. After operations made for tumour, complications occurred more frequently than after interventions because of ulcer. Total mortality was 10.2%. In order to reduce mortality they call attention to the possible prevention of complications and their careful management.

The nucleotide sequence of the gene coding for the 5'-GGCC and 5'-CCGG specific DNA methyltransferase of the Bacillus subtilis phage SPR was determined by the Maxam-Gilbert procedure. Transcriptional and translational signals of the sequence were assigned with the help of S1 mapping and translation in E. coli minicells. The gene codes for a 49 kd polypeptide. The amino acid sequence of the SPR methylase shows regions of homology with the sequence of the 5'-GGCC-specific BspRI modification methylase.

A 2.5 X 10(3) base-pair segment of Bacillus sphaericus R DNA cloned in Escherichia coli has previously been shown to carry the functional BspRI modification methylase gene. The approximate location of the gene on this DNA segment and its direction of transcription were established by subcloning experiments. The nucleotide sequence of the relevant region was determined by the Maxam-Gilbert procedure. An open reading frame that can code for a 424 amino acid protein was found. The calculated molecular weight (48,264) of this protein is in fair agreement with previous estimates (50,000 to 52,000). The synthesis of this protein was demonstrated in E. coli minicells. The initiation point of transcription by E. coli RNA polymerase was localized by in vitro transcription experiments. The open reading frame starts 29 base-pairs downstream from the transcription initiation site and it is preceded by a sequence showing extensive Shine-Dalgarno complementarity. Subcloning experiments and translation in minicells suggest that after removal of this translational initiation site, a secondary start site 29 amino acids downstream can also start translation in E. coli, and this shorter protein retains the methylase activity. The overall base composition of the gene and the codon usage indicate a strong preference for A.T base-pairs.

Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M.FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the (Formula: see text) sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, is completely inhibited by methylation of its recognition site, (Formula: see text), by M.FokIA.

A new plasmid vector, pNS1, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the TcR gene of pNS1 is under the control of the ORpR operator-promoter of phage 434, which is regulated by the repressor gene cI. The cloning sites of pNS1 (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes depressed expression of the TcR gene from pR thus conferring the TcR phenotype on the harboring Escherichia coli strain. The use of pNS1 is facilitated by the presence of another selectable marker, ApR, its small size, and its known nucleotide sequence; no special host strain is required.