Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3.

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene (pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS- and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide (pksM- and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.

Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type background, [corrected] both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. II. Further evidence for a novel function in error-prone repair.

Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage lambda. But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage lambda or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. I. Isolation of uvm mutants and their phenotypical characterization in DNA repair and mutagenesis.

Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.

[Isolation and characterization of Proteus mirabilis mutants deficient in DNA degradation: function of endonuclease I in postmortem DNA degradation]. / Isolierung und Charakterisierung von DNA-Abbaumutanten bei Proteus mirabilis: Funktion der Endonuclease I im postmortalen DNA-Abbau.

DNase deficient mutants of Proteus mirabilis selected for reduced toluene induced DNA degradation were isolated. Their defect in DNA degradation was shown not only after treatment by toluene but also in crude extracts after cell disintegration by ultrasonic and in untreated starved cultures. The degradation mutants behave just as the wild type with respect ot their in vivo functions proffed. The results inidcate that the affected DNase does not have an essential function in vivo but acts in postmortem DNA degradation. Probably the DNase in question concerns the endonuclease I of P. mirabilis described by Goebel and Helinski (1971 a, b).