Cold-sensitive ompB mutants affecting expression of ompC in Escherichia coli K12

The regulatory locus ompB, consisting of 2 genes, ompR and envZ, is required for the expression of ompC and ompF genes encoding the major outer membrane porin proteins OmpC and OmpF in Escherichia coli K12. We utilized localized mutagenesis to isolate cold-sensitive mutants in the ompB operon. The isolated mutants exhibited a cold-sensitive OmpC⁻ phenotype, but remained OmpF⁺. Furthermore, ompC expression was still regulated by medium osmolarity. The cold-sensitive OmpC⁻ phenotype was complemented by plasmids carrying the wild-type ompB operon, but not by plasmids containing either envZ or ompR genes alone. This suggests that the mutations are in the ompB promotor. We show that the mutations can be used to control expression vectors based on the ompC promotor.     

The ompB Operon Partially Determines Differential Expression of OmpC in Salmonella typhi and Escherichia coli

Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi ΔompB strain carrying the ompC-lacZ fusions showed that both S. typhi and E. coli ompC were not regulated by osmolarity when they were under the control of S. typhi ompB. Interestingly, in the same strain, both genes were osmoregulated under E. coli ompB. Surprisingly, in E. coli ΔompB, they were both osmoregulated under S. typhi or E. coli ompB. Thus, the lack of osmoregulation of OmpC expression in S. typhi is determined in part by the ompB operon, as well as by other unknown trans-acting elements present in S. typhi.     

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan^R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.     

DNA sequence of the promoter region of the ompC gene and the amino acid sequence of the signal peptide of pro-OmpC protein in Escherichia coli

The osmoregulated ompC gene of Escherichia coli was cloned and the DNA sequence of a fragment encompassing the promoter region and a portion of the coding region was determined. There were no obvious homologies in the DNA sequence of the promoter regions of the ompC and ompF genes, in contrast to those of the coding regions of the two genes, both of which code for the matrix porins (major outer membrane proteins) and form passive diffusion pores. The amino acid sequence of the signal peptide of pro-OmpC protein was also deduced from the DNA sequence     

Gene fusions to the ptsM/pel locus of Escherichia coli

Summary We have constructed gene fusions between ptsM/pel and lacZ. These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage DNA, as well as that of other lambdoid phages such as Hy-2. Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene, or two genes within the same operon. Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli. This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose. However, we have also identified Tn10 insertion mutations that confer a Man^- phenotype but have no effect on the Pel phenotype. Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes. Both of these genes are involved in mannose uptake. This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of DNA.     

Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2

Summary We have cloned the complete functional ompB locus of Salmonella typhimurium LT-2 into Escherichia coli K-12 using a cosmid vector and in vitro packaging into . The ompB locus of Salmonella was found to complement both envZ and ompR mutations in E. coli as well as an ompR mutation of Salmonella. The ompR part of the ompB locus was further subcloned into the multicopy plasmid pKN410 as a 1.3 kb fragment. This fragment coded for a single 28.5 kd protein corresponding to about 820 bp in length. Furthermore, the OmpR proteins of S. typhimurium and E. coli were shown to be structurally and functionally highly similar.Abbreviations SDS sedium dodecyl sulfate - kb kilobase pairs - bp base pairs - kd kilodaltons     

Identification and Characterization of Two Quiescent Porin Genes, nmpC and ompN, in Escherichia coli BE

The genomic DNA of the B^E strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli B^E.     

Use of lambda vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2

Summary A novel plasmid vector, pAMH70 carrying both the lamB and nusA genes of Escherichia coli K12 was constructed. Introduction of this plasmid into Salmonella typhimurium LT2 renders this bacterium both sensitive to adsorption and able to sustain growth and lysogenization by . Using this strain as a recipient, stable gene fusions to the gene encoding a major outer membrane porin protein OmpC, were constructed with a vehicle placMu. To confirm the actual site of fusions they were genetically mapped and transducing phages carrying the ompC-lacZ fusion were isolated and relysogenized. The fusions were also shown to be to ompC by their regulatory properties.     

Male-specific Y-linked transgene markers to enhance biologically-based control of the Mexican fruit fly, <Emphasis Type="Italic">Anastrepha ludens</Emphasis>(Diptera: Tephritidae)

Background

Reliable marking systems are critical to the prospective field release of transgenic insect strains. This is to unambiguously distinguish released insects from wild insects in the field that are collected in field traps, and tissue-specific markers, such as those that are sperm-specific, have particular uses such as identifying wild females that have mated with released males. For tephritid fruit flies such as the Mexican fruit fly, Anastrepha ludens, polyubiquitin-regulated fluorescent protein body markers allow transgenic fly identification, and fluorescent protein genes regulated by the spermatocyte-specific β2-tubulin promoter effectively mark sperm. For sterile male release programs, both marking systems can be made male-specific by linkage to the Y chromosome.

Results

An A. ludens wild type strain was genetically transformed with a piggyBac vector, pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3}, having the polyubiquitin-regulated EGFP body marker, and the β2-tubulin-regulated DsRed.T3 sperm-specific marker. Autosomal insertion lines effectively expressed both markers, but a single Y-linked insertion (Y^EGFP strain) expressed only PUbnlsEGFP. This insertion was remobilized by transposase helper injection, which resulted in three new autosomal insertion lines that expressed both markers. This indicated that the original Y-linked Asβ2tub-DsRed.T3 marker was functional, but specifically suppressed on the Y chromosome. The PUbnlsEGFP marker remained effective however, and the Y^EGFP strain was used to create a sexing strain by translocating the wild type allele of the black pupae (bp⁺) gene onto the Y, which was then introduced into the bp^- mutant strain. This allows the mechanical separation of mutant female black pupae from male brown pupae, that can be identified as adults by EGFP fluorescence.

Conclusions

A Y-linked insertion of the pBXL{PUbnlsEGFP, Asβ2tub-DsRed.T3} transformation vector in A. ludens resulted in male-specific expression of the EGFP fluorescent protein marker, and was integrated into a black pupae translocation sexing strain (T(Y^EGFP/bp⁺), allowing the identification of male adults when used in sterile male release programs for population control. A unique observation was that expression of the Asβ2tub-DsRed.T3 sperm-specific marker, which was functional in autosomal insertions, was specifically suppressed in the Y-linked insertion. This may relate to the Y chromosomal regulation of male-specific germ-line genes in Drosophila.

     

Characterization of bacteriophages specificity for antibiotic-resistant <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

This study aimed to propose a new approach to understand the binding interaction between bacteriophages and antibiotic-resistant Salmonella typhimurium. The antibiotic susceptibilities of S. typhimurium strains were determined using a broth dilution method. The phage adsorption rates were determined to evaluate the lytic ability of bacteriophages against S. typhimurium strains. Bacterial outer membrane proteins and lipopolysaccharide (LPS) were analyzed to evaluate the antibiotic-induced alteration in bacterial cell surface receptors. The relative expression levels of outer membrane-, flagella-, porin-, and O-antigen-related genes were estimated using a qPCR assay. Compared to ST^WT, the ST^CIP exhibited a reduced susceptibility to cefotaxime (32-fold), ciprofloxacin (32-fold), meropenem (16-fold), and norfloxacin (64-fold). PBST35 produced adsorption rates of 82–95% at ST^WT, ST^CIP, and ST^CCARM within the first 10 min of infection. Compared to ST^WT, ST^CIP exhibited less protein bands between 24 and 36 kDa, corresponding to the low adsorption rates of P22 and PBST10. The relative expression levels of outer membrane-related genes (btuB, ompC, and tolC), flagellar-related genes (fliC, fljB, and fliK), porin-related gene (fhuA), and O-antigen-related genes (rfaL) were decreased in ST^CIP. The alteration in bacteriophage-binding receptors resulted in the low adsorption rate. Our findings provide new insights for effective treatment against antibiotic-resistant bacteria. The results would help to develop new therapeutic strategy as a prospective alternative control of antibiotic-resistant bacteria.     

Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora

Summary A locus, ompRS, controlling synthesis of outer membrane proteins was cloned from Erwinia carotovora subsp. carotovora (Ecc) by complementation of an Escherichia coli ompR—envZ mutant. The Ecc ompRS locus was both structurally and functionally similar to the ompR—envZ operon controlling porin gene expression in E. coli as shown by DNA hybridization and complementation of E. coli ompR and envZ mutants. Furthermore, introduction of ompRS into E. coli (ompR—envZ) strains restored the osmoregulation of the major outer membrane protein genes ompC and ompF Maxicell analysis of ompRS-carrying plasmids suggested that proteins similar in size to the E. coli ompR and envZ gene products were encoded by the Ecc ompR and ompS genes, respectively. Introduction of an ompRS transposon mutant onto the Ecc chromosome by marker exchange mutagenesis showed that ompRS is essential for production of a major outer membrane porin in Ecc. This mutational defect could be complemented by clones carrying Ecc ompRS or E. coli ompR envZ. The lack of the porin did not, however, compromise the virulence of these Ecc mutants.     

Insertional Editing of Mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G·C base pair. Uridine insertions have been identified in the T loop of tRNA^Lys from Didymium and tRNA^Glu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTΨC (Ψ = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.