Nitrofurantoin-resistant mutants of Escherichia coli: isolation and mapping

Summary Mutants of Escherichia coli resistant to nitrofurantoin have been isolated. The mutations, designated nfnA and nfnB were introduced individually into a multiply auxotrophic E. coli F^– strain and mapped by conjugation and transduction. nfnA is located at 79.8 min and nfnB at 13.0 min on the E. coli chromosome.     

Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer

Summary A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E. coli K12 strains. No fragments capable of complementing leuA or leuD mutations were identified. Conjugal transfer of the LeuB/leuC genes from Bacillus subtilis into two different Leu^- C. acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC. Inheritance of the cointegrate plasmid restored one of the auxotrophic C. acetobutylicum strains to prototrophy. The second strain remained Leu^-.     

General transducing phages like Salmonella phage P22 isolated using a smooth strain of Escherichia coli as host

A smooth colony strain, resistant to phages λ and P22, was isolated from sewage and identified as Escherichia coli (strain H). Four temperate phages plaquing on strain H were isolated from sewage. The archetype, HK620, does not plaque on strains C and K12 of E. coli nor on the LT2 strain of Salmonella enterica. Bacterial mutants resistant to a clear plaque mutant of HK620 produce rough colonies. Some are also galactose-negative, a few are histidine auxotrophs, and most show sensitivity to λ. HK620 can transduce a wide variety of auxotrophic mutants of E. coli H to prototrophy. It can recombine with λ but its virions resemble those of P22.     

Production of lysine by mutants of escherichia coli K 12 in a medium with lactose

In an effort to use whey for lysine production, we isolated from a β-galactosidase-hyperproducing strain of E. coli K 12 multiple mutants – auxotrophic, regulatory and penicillin-resistant. These mutants exhibited for the most part a high reversion rate but some of them produced about 2 mg/ml lysine in an enriched fermentation medium.     

The Effect of Red and Infrared Light on the Growth of Escherichia coli and the Production of the Recombinant Protein Barstar

Incoherent red and infrared low-intensity light enhanced the growth of the auxotrophic strain Escherichia coli AD494(DE3)pLysS and the production of the recombinant polypeptide barstar. Illumination also stimulated the growth of nonrecombinant E. coli cells.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.     

Isolation and characterization of a ribonuclease III deficient mutant of Escherichia coli

Summary A mutant of E. coli K 12 AB301 RNAase ₁₉ ^- , selected for its inability to degrade double-stranded RNA, has been isolated and shown to have less than 1% of RNAase III-activity related to the parental strain.Abbreviations TCA trichloracetic acid - RF replicative form of phage-RNA Enzymes Lysozyme (E.C. 3.2.1.17) - RNAase (E.C. 2.7.7.16) - DNAase (E.C. 3.1.4.5)     

Occurrence and genetic association of selected virulence factors in clinicalEscherichia coli isolates

Occurrence ofcnf1+ E. coli pathogenic strains among extraintestinalE. coli isolates was evaluated to explain an impact of cytotoxic necrotizing factor type 1 (CNF1) in human infections. A total of 120E. coli isolates were characterized for presence of virulence factorscnf1- andpap- specific sequences by PCR, and the production of α-hemolysin using blood agar-plate test. Different association patterns among the detected virulence factors were obtained by comparison of various groups of clinicalE. coli isolates. These differences probably reflect a potential impact of CNF1 in the colonization of vaginal environment.     

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn²⁺ (48 mM), Cu²⁺ (12 mM), Ni²⁺ (12 mM), chloramphenicol (50 μg/ml), and tetracycline (25 μg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn²⁺, 12 mM Cu²⁺, and 12 mM Ni²⁺, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell. Received: 5 April 2002 / Accepted: 5 July 2002     

Synthetic mutualism in engineered E. coli mutant strains as functional basis for microbial production consortia

In nature, microorganisms often reside in symbiotic co-existence providing nutrition, stability, and protection for each partner by applying “division of labor.” This principle may also be used for the overproduction of targeted compounds in bioprocesses. It requires the engineering of a synthetic co-culture with distributed tasks for each partner. Thereby, the competition on precursors, redox cofactors, and energy—which occurs in a single host—is prevented. Current applications often focus on unidirectional interactions, that is, the product of partner A is used for the completion of biosynthesis by partner B. Here, we present a synthetically engineered Escherichia coli co-culture of two engineered mutant strains marked by the essential interaction of the partners which is achieved by implemented auxotrophies. The tryptophan auxotrophic strain E. coli ANT-3, only requiring small amounts of the aromatic amino acid, provides the auxotrophic anthranilate for the tryptophan producer E. coli TRP-3. The latter produces a surplus of tryptophan which is used to showcase the suitability of the co-culture to access related products in future applications. Co-culture characterization revealed that the microbial consortium is remarkably functionally stable for a broad range of inoculation ratios. The range of robust and functional interaction may even be extended by proper glucose feeding which was shown in a two-compartment bioreactor setting with filtrate exchange. This system even enables the use of the co-culture in a parallel two-level temperature setting which opens the door to access temperature sensitive products via heterologous production in E. coli in a continuous manner.     

A temperature sensitive nonsense mutation affecting the synthesis of a major protein of Escherichia coli K12

Summary A temperature sensitive nonsense (TSN) mutant of E. coli K12 has been isolated in which a major bacterial protein is not synthesized at 42° C. This protein is found in the parental strain at 42° C and in cells rendered temperature resistant due to the insertion of a number of different nonsense suppressors or the normal allele of the mutant locus.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

Characterization of an iron sensitive Mud1 mutant in E. coli lacking the ribonucleotide reductase subunit B2

The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air. Under anaerobic conditions normal growth in the presence of iron was observed. The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli. By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2. The mutation could be complemented by the cloned nrdB gene. Up to now it was assumed that E. coli possesses only one enzyme for the synthesis of deoxyribunucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB. The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit. Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides an additional pathway for the synthesis of deoxyribonucleotides is postulated.     

Front Cover Image,Volume 116, Number 12, December 2019

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 10⁷ plaque-forming units/ml and produce GFP with a yield of up to 30 μg/10⁹ colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.     

Lethal and mutagenic lesions induced by ionizing radiations inE. coli and DNA strand breaks

Summary The inactivation doses forE. coli exposed to alpha particles and protons of different LETs and to gamma rays have been measured. Strains derived fromE. coli B/r showed a maximum sensitivity at LETs of 30 keV/ whilst B_s–1 and other strains known to be deficient in repair capacity had sensitivities which decreased monotonically with increasing LET. These results can be interpreted in terms of two types of lethal damage to the bacterial genome. Damage of type 1 affects only one strand of the DNA macromolecule and is partially reparablein vivo whilst damage of type 2, inflicted by one track intersection of the DNA, is irreparable. The identity of both types of damage is uncertain but type 1 probably gives rise to a lesion recognizablein vitro as a single strand break. Type 2 damage probably corresponds to double strand scission of DNA as observedin vitro.Mutations to prototrophy of three auxotrophic strains ofE. coli are induced with an effectiveness which decreases steadily with increasing LET. This form of LET dependence implies that these mutations involve damage to one target only, probably one strand of the DNA duplex.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin