Genome-wide transposon mutagenesis of Borrelia burgdorferi for identification of phenotypic mutants

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.     

Site-directed mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium

Site-specific mutagenesis has been used to prepare two mutant forms of the alpha subunit of tryptophan synthase from Salmonella typhimurium in which either cysteine-81 or cysteine-118 is replaced by a serine residue. These mutant proteins are potentially useful for x-ray crystallographic studies since a heavy metal binding site is specifically eliminated in each mutant. The purified mutant proteins are fully active in four reactions catalyzed by the wild type alpha 2 beta 2 complex of tryptophan synthase. However, the mutant alpha 2 beta 2 complexes dissociate more readily and are less heat-stable than the wild type alpha 2 beta 2 complex. Thus, cysteine-81 and cysteine-118 of the alpha subunit serve structural but not functional roles.     

Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants

To study the role of the hinge region in nisin and to obtain mutants that exhibit altered or new biological activities and functional properties, we changed certain amino acids in the hinge region by performing site-directed mutagenesis with the nisinZ structural gene (nisZ). The results showed that the nisinZ mutants had decreased antimicrobial activities against Micrococcus flavus NCIB8166 and Streptococcus thermophilus. Interestingly, compared with wild nisinZ, mutant N20K nisinZ and M21K nisinZ displayed antimicrobial activity against gram-negative Shigella, Pseudomonas and Salmonella; and they had a higher solubility than wild-type nisinZ. At pH 8, the solubilities of N20K nisinZ and M21K nisinZ were, respectively, three-fold higher and five-fold higher than that of nisinZ. Mutant N20Q nisinZ and M21G nisinZ were considerably more stable than nisinZ at higher temperatures and neutral or alkaline pH. These mutants provided information that the central hinge region in nisinZ plays an important role in providing the conformational flexibility required for the antimicrobial activity on the membrane. Our finding documented that it may well be worth considering the construction of the new nisin mutants with changed inhibitory activity against a wide range of gram-negative bacteria and the improvement of functional properties by site-directed mutagenesis.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

Recombination-deficient mutants of colicinogenic Salmonella typhimurium detected by their failure to produce colicin

Survivors of nitrosoguanidine-treated cultures of a colicinogenic strain of Salmonella typhimurium were tested for spontaneous production of colicin E1. Of about 1,000 colonies tested, 13 produced no (or very narrow) colicin zones. Four of these isolates proved to be more sensitive to ultraviolet (UV) light, X rays, and methyl methane sulfonate than the parent strain and did not show enhanced production of colicin when treated with mitomycin C (which acts as an inducer on wild-type cells). Further studies showed that these isolates were of two classes. Three mutants were extremely sensitive to UV, failed to show spontaneous release of two temperate phages, and were infertile as recipients in transduction or in an Hfr cross although they accepted an F' factor normally. These independently isolated mutants were inferred to be recombination-deficient; one of them had the additional property of increased spontaneous mutability at two loci. The other colicin-nonreleasing isolate was only moderately sensitive to UV, showed enhanced spontaneous release of two temperate phages, and was of approximately normal fertility as a recipient in transduction or conjugation.     

Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils

Aims:  To evaluate factors potentially contributing to the long-term persistence of Salmonella enterica serovar Enteritidis phage type (PT) 30 in an almond orchard. Methods and Results:  Surface and subsurface soil temperatures, and air temperatures in a radiation shelter, were recorded during a 12-month period, and were used to identify relevant storage temperatures (20 or 35°C) for microcosms of two different soil types (clay and sandy loams) with moisture levels near saturation or near field capacity. Salmonella Enteritidis PT 30 was inoculated into the microcosms at 6 log CFU g⁻¹ dry weight. Between 14 and 180 days of incubation, counts of S. Enteritidis PT 30 decreased rapidly at 35°C and were significantly different (P < 0·05) from counts at 20°C, regardless of the soil type or moisture level. Salmonella was detected by enrichment of 10-g samples from all microcosms after 180 days of incubation at 20°C, but from none of the microcosms held at 35°C. To measure the potential for the growth of S. Enteritidis PT 30 in clay loam soil, an aqueous extract of almond hulls (containing 1·6% mono and disaccharides) or equivalent volume of water was added 7 days after inoculation. Significant (P < 0·05) growth of S. Enteritidis PT 30 was observed within 8 or 24 h of adding hull extract, but not water, to soil. Conclusions:  Opportunities may exist for S. Enteritidis PT 30 to survive for an extended time in almond orchard soils and to grow in these soils where hull nutrients are released. Significance and Impact of the Study:  Temperature has a significant impact on the long-term survival of S. Enteritidis PT 30 in soil, and nutrients leached from almond hulls may result in Salmonella growth. These factors should be considered in the design of Good Agricultural Practices for almonds.     

Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase

The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2Delta)) having two (3R)-hydroxyacyl-CoA dehydrogenases with different substrate specificities has been purified and crystallized as a recombinant protein. The expressed construct was modified so that a stabile, homogeneous protein could be obtained instead of an unstabile wild-type form with a large amount of cleavage products. Cubic crystals with unit cell parameters a=74.895, b=78.340, c=95.445, and alpha=beta=gamma=90 degrees were obtained by using PEG 4000 as a precipitant. The crystals exhibit the space group P2(1)2(1)2(1) and contain one molecule, consisting of two different (3R)-hydroxyacyl-CoA dehydrogenases, in the asymmetric unit. The crystals diffract to a resolution of 2.2A at a conventional X-ray source.     

Association of attenuated mutants of Salmonella enterica serovar Enteritidis with porcine peripheral blood leukocytes

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.     

Site-directed mutagenesis of the reactive center (serine 394) of antithrombin III

Human antithrombin III (AT) shares significant sequence homology and a common inhibitory mechanism with the serine protease inhibitor (serpin) superfamily. AT has a reactive site in which the P1 residue is primarily responsible for protease specificity. The P1' residue, almost invariably serine, is critical in the inactive natural variant AT-Denver, which has a leucine substitution in that position (Stephens, A.W., Thalley, B.S., and Hirs, C.H.W. (1987) J. Biol. Chem. 262, 1044-1048). In the present study site-directed mutagenesis was used to generate eight variants with altered P1' residues. All were secreted efficiently by COS cells transiently transfected with the AT cDNA in a eukaryotic shuttle vector. All variants also bound heparin as effectively as wild-type AT. Variants were grouped into three categories with respect to thrombin-AT complex formation: 1) no detectable inhibitory activity (proline, methionine); 2) low activity (cysteine, valine, leucine); and 3) near normal activity (glycine, alanine, threonine). The leucine variant, which is in the low activity group, exhibited the same physical and functional properties as AT-Denver. We conclude that the serine hydroxyl is not critical for functional activity and that there is a side chain size optimum which is modulated by hydrophobic effects.     

Development of an efficient in vivo system (Pjunc-TpaseIS1223) for random transposon mutagenesis of Lactobacillus casei

The random transposon mutagenesis system P(junc)-TpaseIS(1223) is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the P(junc) sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation.     

Novel motility mutants of synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis

We screened for transposon-generated mutants of Synechocystis sp. strain PCC 6803 that exhibited aberrant phototactic movement. Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes. These novel genes and their possible roles in motility are discussed.     

Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima)

In order to make cost-effective bioethanol from dynamic lignocellulosic material, we require potentially acting and stable cellulolytic enzymes. In our investigation, the hyperthermostable endoglucanase Cel5A from Thermotoga maritima was subjected to site-directed mutagenesis and carbohydrate-binding module (CBM) engineering. For this purpose, amino acids around the active-site region were targeted. Results indicated that five single mutants showed a shift in optimal pH from 5 to 5.4. The N147E mutant displayed 10% higher activity than native Cel5A. Domain engineering was performed with fungal and bacterial CBM. In addition, CBM1 from (CBHII) Trichoderma reesei and CBM6 from Clostridium stercorarium xylanase A were fused with Cel5A. Both the CBM-engineered Cel5A showed 14-18-fold higher hydrolytic activity towards Avicel. Immuno-gold labeling assay of engineered enzymes further indicated the relativity that exists between binding ability and activity.     

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.     

Isolation of Gln- mutants through transposon, Tn917, mutagenesis in Bacillus brevis.

Transposon, Tn917, carried on pTV1 plasmid has been used successfully to mutagenise Bacillus brevis. The transposon showed preference for insertion at an "aro" site. A second insertional event after elimination of the preferred site with ethidium bromide/acridine orange treatment has permitted isolation of Gln- mutants in B. brevis.     

Antimutagenic effects of chemicals on mutagenesis resulting from excision of a transposon in Salmonella typhimurium

We have found that a temperature-sensitive mutation in the polA gene of Salmonella typhimurium strain LT2 causes precise excision of transposon Tn10 to occur at significantly increased frequencies in cells incubated at the restrictive temperature. In our experiments, precise excision from a site in the tryptophan operon was measured by determining the frequency of reversion of the auxotrophic trp1014::Tn10 polA7 strain to prototrophy on defined medium containing a trace amount of broth. Because the yields of revertants at 37 degrees C were of the order of 200 colonies per plate, it was possible to measure the effects of chemical inhibitors on the processes involved in precise excision. We now report that all of the DNA-repair inhibitors we have studied (caffeine, ethionine, acriflavine, procaine and cinnamaldehyde) are effective inhibitors of precise excision of Tn10, and can therefore be defined as antimutagens.