Minicell Expression Research Articles

Genetics of alkane oxidation by Pseudomonas oleovorans.

Many Pseudomonads are able to use linear alkanes as sole carbon and energy source. The genetics and enzymology of alkane metabolism have been investigated in depth for Pseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid. The so-called alk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis. This has led to the identification and characterization of of the alkBFGHJKL and alkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives. These then enter the beta-oxidation-cycle, and can be utilized as carbon- and energy sources. Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized. The alkane-oxidizing enzymes used by some of these strains (e.g. two P. aeruginosa strains, a P. denitrificans strain and a marine Pseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid.

Genetic Organization of the Conjugal DNA Processing Region of the IncW Plasmid R388

The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOB w) has been analyzed by Tn5tacl insertion mutagenesis, genetic complementation and DNA sequencing. Three genes ( trwA, trwB and trwC) were mapped within MOB w. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13·5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases, It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.

Cloning and sequence analysis of an Escherichia coli gene conferring bicyclomycin resistance

We have cloned and sequenced DNA from Escherichia coli that, when present in a high-copy-number plasmid, confers resistance to the diketopiperazine antibiotic, bicyclomycin (Bc). The DNA includes a 378-amino-acid open reading frame (ORF), disruption of which results in the loss of Bc resistance. This ORF contains the Bc R gene. Studies using the minicell expression system reveal that a polypeptide of 31 kDa is produced from this cloned region. The ORF maps at 47.1 min on the E. coli genome map. Sequence comparison between the translated ORF and a protein database reveal between 26.5 and 23.4% aa sequence homology to bacterial transmembrane (TM) proteins including those mediating chloramphenicol (Cm) and tetracycline (Tc) resistance and an arabinose-proton symport protein. Sequence analysis using the Diagon program showed the Bc R gene product (BcR) had homology with the N-terminal regions of the Cm R and Tc R-encoded proteins and weak N-terminal homology with the arabinose-proton symport protein. Hydropathy profiles of the BcR protein and Cm R products show a striking similarity, both having twelve predicted TM domains.

A Salmonella dublin virulence plasmid locus that affects bacterial growth under nutrient-limited conditions.

This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal-medium containing various sole carbon/energy sources, unlike the wild-type and plasmid-cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the phenotypic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal-medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.

Cloning and DNA sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000

The complete nucleotide sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000 has been determined. Computer analysis of this 4821-bp plasmid for protein coding regions, based on mycobacterial codon usage preferences, reveals the presence of two putative protein coding regions immediately downstream from typical mycobacterial promoter and ribosome binding sites. Both open reading frames, ORF1 and ORF2, produced proteins with the predicted respective sizes previously shown in minicell expression experiments [A. Labidi et al. (1985) FEMS Microbiol. Lett. 30, 221–225]. ORF1 encodes a putative 20-kDa basic protein with characteristics of a DNA binding protein involved in plasmid DNA replication. ORF2 encodes a 67-kDa protein with an amino-terminal sequence suggestive of a transported protein and a possible transmembrane anchor near its carboxyl-terminal. The current sequence and its analysis are more consistent with the minicell expression experiments than the previously published sequence of the pAL5000 plasmid [J. Rauzier et al. (1988) Gene 71, 315–321].

Molecular cloning, partial purification, and characterization of a haemin‐binding lipoprotein from Haemophilus influenzae type b

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.

Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-related with its S. typhi counterpart.

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that it is a polypeptide of Mr 12,000 with 99 amino acid residues and contains a potential helix-turn-helix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.

Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.

Aeromonas salmonicida Plasmids: Plasmid-directed Synthesis of Proteins in vitro and in Escherichia coli Minicells

Plasmid DNAs from 25 Aeromonas salmonicida strains (14 ‘typical’ and 11 so-called ‘atypical’ strains) were analysed using two plasmid isolation techniques in conjunction with restriction enzyme digestion and agarose gel electrophoresis. ‘Atypical’ strains carried two to four plasmid species, which were different from the plasmids carried by ‘typical’ strains, and corresponded to isolate source and biotype, suggesting that plasmid content may be a useful epidemiological marker for ‘atypical’ A. salmonicida. The ‘typical’ group was extremely homogeneous in plasmid content. In addition to a single large (70–145 kb) structurally related plasmid species, all strains carried a highly conserved group of three low-M r plasmids (pAsa1, 5·0 kb; pAsa2, 5·2 kb; pAsa3, 5·4 kb). The pAsa plasmids had different restriction maps, and directed the synthesis of four polypeptides when assayed in vitro using a transcription/translation system and [3 5S]methionine radiolabelling. A partially conserved 6·0 kb plasmid directed synthesis of an additional polypeptide. Total cellular plasmid DNA from virulent ‘typical’ strain A. salmonicida A449 was also cloned in Escherichia coli, using the vector pBR322. When analysed using a minicell expression system, 17 plasmid-encoded proteins, ranging in size from 12 to 90 kDa, were identified, including two putative exported proteins, and a chloramphenicol acetyltrans-ferase which was encoded by the 145 kb conserved, non-conjugative, plasmid.

Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme.

A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Escherichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.

Ferric-coprogen receptor FhuE of Escherichia coli: processing and sequence common to all TonB-dependent outer membrane receptor proteins

Iron transport via siderophores requires outer membrane receptor proteins and the TonB protein. The FhuE protein of Escherichia coli functions as the receptor for ferric coprogen and ferric-rhodotorulic acid. A chromosomal DNA fragment bearing the fhuE gene was cloned into pACYC184. The gene was localized by insertion mutagenesis by using the transposon Tn1000. Expression in minicells revealed a FhuE precursor with an apparent molecular weight of 82,000 and a FhuE protein with a molecular weight of 76,000. The transcription polarity of the fhuE gene was deduced from the size of truncated polypeptides derived from Tn1000 insertions, which were mapped by restriction analysis. The processing of truncated precursors that were synthesized by insertion mutants was strongly reduced even when the insertion site was close to the carboxy terminus of the FhuE protein. It is concluded that either the efficient insertion of proFhuE into the cytoplasmic membrane or the rate of cleavage of the signal peptide requires a particular conformation of the proFhuE protein, which is only formed by the complete primary structure. The amino-terminal amino acid sequence deduced from the nucleotide sequence was confirmed by gas-phase sequencing of the precursor and the mature form, which were separated by electrophoresis on polyacrylamide gels. The precursor contained an unusually long signal peptide of 36 amino acids. The amino-terminal end of the mature form contained the sequence Glu-Thr-Val Ile-Val. A pentapeptide starting with either Glu or Asp, followed by Thr, and two uncharged residues ending with Val were found in all outer membrane receptor proteins that were constituents of TonB-dependent transport systems.

Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins.

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.

Biosynthetic arginine decarboxylase in Escherichia coli is synthesized as a precursor and located in the cell envelope.

The biosynthetic form of arginine decarboxylase (ADC) catalyzes the synthesis of agmatine, a precursor of putrescine, in Escherichia coli. Selective disruption of the cell envelope and an assessment of ADC activity or immunoprecipitable ADC in various fractions demonstrated its location between the cytoplasmic membrane and peptidoglycan layer. Expression in minicells of the speA gene encoding ADC resulted in the production of two immunoprecipitable species (74 and 70 kilodaltons). Studies in vivo with a pulse and chase of radiolabeled amino acid into the two species suggest a precursor-product relationship. This relationship was corroborated by demonstrating the accumulation of the 74-kilodalton species in a strain of E. coli unable to process signal sequences. Peptide mapping experiments with V8 protease, trypsin, and alpha-chymotrypsin demonstrated that the two species of ADC were very similar except for a minor difference. These data were used to substantiate the compartmentalization hypothesis as to how exogenous arginine can be channeled preferentially into putrescine.

Cryptic plasmid of Neisseria gonorrhoeae: complete nucleotide sequence and genetic organization.

The naturally occurring cryptic plasmid pJD1 of Neisseria gonorrhoeae is 4,207 base pairs long and is found in about 96% of gonococcal strains. The total probable coding capacity of pJD1 was determined from the complete nucleotide sequence by using computational probes to identify open reading frames with similar codon usage and by screening for the presence of ribosomal binding sites before the start codons. Candidates for promoters and terminators were also found in the sequence. Based on these findings, we propose a model for the genetic organization of the plasmid. The model predicts two transcriptional units, each composed of five compactly spaced genes. A promoter of one of the transcripts was shown to function in Escherichia coli, and the products of three of the five genes in this operon were identified in minicell expression experiments. Of these, the cppA gene encoded a 9-kilodalton protein, and the cppB and cppC genes both coded for 24-kilodalton proteins. No expression of the other transcriptional unit was detected, but two genes in this operon were expressed in minicells when transcribed from an E. coli promoter. The experimental data were consistent with the model.

Thermoregulated expression of a cloned congo red binding activity gene ofShigella flexneri

Abstract A 2.15-kb Pst I fragment of the 140-megadalton (MDa) plasmid of Shigella flexneri which encodes Congo red (CR) dye binding activity was isolated by recombinant techniques. The recombinant plasmid was shown to encode the structural gene for CR binding but failed to restore the ability of noninvasive strains to penetrate HeLa cell monolayers. A partial restriction map was constructed and polypeptides specified by the 140-MDa insert DNA identified in a minicell system. The putative polypeptide responsible for CR binding activity was identified by its thermoregulated expression in minicells. The M r of the putative CR binding protein was 24000.

Thermoregulated expression of a cloned congo red binding activity gene of Shigella flexneri

A 2.15-kb Pst I fragment of the 140-megadalton (MDa) plasmid of Shigella flexneri which encodes Congo red (CR) dye binding activity was isolated by recombinant techniques. The recombinant plasmid was shown to encode the structural gene for CR binding but failed to restore the ability of noninvasive strains to penetrate HeLa cell monolayers. A partial restriction map was constructed and polypeptides specified by the 140-MDa insert DNA identified in a minicell system. The putative polypeptide responsible for CR binding activity was identified by its thermoregulated expression in minicells. The Mr of the putative CR binding protein was 24000.

Globoside-specific adhesins of uropathogenic Escherichia coli are encoded by similar trans-complementable gene clusters.

Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells. For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA. Instead, two other pap genes papF and papG are specifically required for globoside binding (F. P. Lindberg et al., EMBO J. 3:1167-1173, 1984). By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E. coli isolates. Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG). The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.

Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes.

Region III flagellar genes in Escherichia coli are involved with the assembly and rotation of the flagella, as well as taxis. We subcloned the flaB operon from a lambda fla transducing phage onto plasmid pMK2004. Two additional genes were found at the flaB locus, and we subdivided the flaB gene into flaB1, flaBII, and flaBIII. The cheY suppressor mutations which have previously been mapped to flaB were further localized to flaB11 (Parkinson et al., J. Bacteriol. 155:265-274, 1983). Until now, gene product identification has not been possible for these genes because of their low levels of gene expression. Overexpression of the flagellar genes was accomplished by placing the flaB operon under the control of the lacUV5 or tac promoters. Plasmid-encoded proteins were examined in a minicell expression system. By correlating various deletions and insertions in the flaB operon with the ability to complement specific flagellar mutants and code for polypeptides, we made the following gene product assignments: flaB 1, 60 kilodaltons; flaB 11, 38 kilodaltons; flaB111, 28 kilodaltons; flaC, 56 kilodaltons; fla0, 16 kilodaltons; and flaE, 54 kilodaltons.

A cluster of five genes specifying the aerobactin iron uptake system of plasmid ColV-K30.

The genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30, cloned as recombinant plasmid pABN1, were mapped by insertional inactivation using Tn1000 (gamma delta). Sites of insertion resulting in loss of aerobactin biosynthesis spanned ca. 5.5 kilobase pairs of cloned ColV-K30 DNA contiguous with a 2-kilobase-pair region in which transposon insertion resulted in loss of the outer membrane ferric-aerobactin receptor protein. Translation products of plasmid pABN1, and of subclones specifying siderophore biosynthesis alone or receptor activity alone, were analyzed by using the maxicell and minicell expression system. Four polypeptides (Mr = 62,000, 35,000, 45,000, and 50,000) are required for biosynthesis of aerobactin. A fifth product (Mr = 74,000) of plasmid pABN1 represents the outer membrane receptor protein. The linear order of genes for these polypeptides was determined by comparing translation products of a series of smaller derivative plasmids and of a number of mutant plasmids carrying Tn1000 at known locations.