The sigma-like product of sporulation gene spoIIAC of Bacillus subtilis is toxic to Escherichia coli

Summary The amino-acid sequence deduced from the nucleotide sequence of the spoIIAC gene of Bacillus subtilis has been shown to be homologous to that of the sigma subunit of the Escherichia coli RNA polymerase (Errington et al. 1985). I now describe results that indicate that this gene can be cloned in E. coli only under conditions in which it is not expressed.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.This paper is affectionately dedicated to Prof. John L. Ingraham     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Cloning of the gene coding for chitobiase ofSerratia marcescens

Summary A λ phage DNA library ofSerratia marcescens was constructed and a clone carrying the gene coding for chitobiase (E.C.3.2.1.29) was isolated and characterized. Deletion analysis limited the cloned region to 4.5 kb that is capable of efficient expression of chitobiase.Escherichia coli cells harboring a plasmid carrying the cloned gene express chitobiase constitutively. The molecular weight of the protein is about 95000 daltons. In exponentially growingE. coli cells the chitobiase enzyme was found to be secreted into the periplasm.     

Secondary structure of the autoregulatory mRNA binding site of ribosomal protein L1

Summary The secondary structure of the autoregulatory mRNA binding site of Escherichia coli ribosomal protein L1 has been studies using enzymatic methods. The control region of the E. coli L11 operon was cloned into a vector under control of the Salmonella phage SP6 promoter, and RNA transcribed using SP6 RNA polymerase. The secondary structure of this RNA was probed using structure-specific nucleases, and by comparison of the data with computer predictions of RNA folding, secondary structural features were deduced. The proposed model is consistent with elements of some previously proposed models, but differs in other features. Finally, secondary structure information was obtained from two mutant mRNAs and the structural features correlated with observed phenotypes of the mutants.Abbreviations MB mung bean nuclease - V₁ cobra venom nuclease - sss single-strand-specific - dss double-strand-specific     

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21^Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21^Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21^Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described. Correspondence to: G. Reipen     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

Cloning and expression of <Emphasis Type="Italic">Bacillus thuringiensis cry</Emphasis>11 crystal protein gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The six most toxic Pakistani isolates of Bacillus thuringiensis (SBS Bt-23, 29, 34, 37, 45 and 47), which were previously characterized for their toxicity against larvae of mosquito, Anopheles stephensi, and the presence of cry4 gene, were used for cry11 (cry4D) gene amplification. A 1.9-kb DNA fragment of cry11 gene was PCR-amplified, cloned in expression vector pT7-7, and then used for transformation of E. coli BL21C. The optimum expression was obtained with 1 mM IPTG at 37°C for 3 h. This gene showed different percentage homologies at protein level with scattered mutations in the toxic region. Biotoxicity assay of recombinant protein showed that Cry11 of SBS Bt 45 (DAB Bt 5) was the most toxic protein against third instar larvae of mosquito, A. stephensi, and has potentiality of a bioinsecticide against mosquitoes.     

Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20–27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD. Received: 13 July 1996 / Revised: 6 August 1996     

Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

Summary The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific d-phenylalanine dependent ATP-³²PP_i and 2deoxy ATP-³²PP_i exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector.     

An insertion sequence unique to Frankia strain ArI5

At the genetic level, understanding of symbiotic nitrogen fixation by Frankia is limited to nif functions that are highly conserved among all organisms. The genetics and biochemistry of nodulation are largely unexplored because of a complete lack of genetic tools. In other bacteria, mobile genetic elements such as insertion sequences (IS) and transposons are commonly used to create mutations and insert new genetic material. We have characterized a 4 kbp segment of DNA from Frankia strain ArI5 that has the hallmarks of a mobile genetic element, inverted repeats flanking a gene encoding a transposase. There are at least six copies of this element in strain ArI5 but none in either strain CcI3 or CpI1. The inverted repeats are 17 nt long and separated by 2156 bp. Within that region are two, overlapping ORFs that each encode a transposase. RT-PCR analysis of RNA from Frankia ArI5 cells conclusively demonstrates the expression of one transposase gene and suggests that both may be transcribed. Numerous attempts to clone the intact IS in E. coli were unsuccessful suggesting that the element may be unstable in this context. A clone containing the complete IS was constructed in E. coli then modified by insertion of the kanamycin (KAN) resistance gene from Tn5. A fragment of DNA including the inverted repeats, transposase genes and KAN gene, was transferred to the suicide vector pJBSD1. The construct, pFRISK, was transformed into E. coli to search for transposition events.     

Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12

Summary The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined. This region was found to contain the dnaY gene encoding a transfer RNA. The curved DNA structure was demonstrated to be located just upstream of the dnaY promoter. The results of sequencing further revealed that the int gene of a cryptic prophage, qsr, which has been shown to be present in the E. coli genome, is located next to the dnaY gene.