Structure of the Pseudomonas putida alkBAC operon. Identification of transcription and translation products

The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment. We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops. Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells. Using a spectrum of overlapping subclones, six different proteins were identified. Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively. The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody. The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex. The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

Physical and genetic characterization of the glucitol operon in Escherichia coli.

The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A. Sancar and W. D. Rupp, Proc. Natl. Acad. Sci. USA 76:3144-3148, 1979). We subcloned the gut operon by using paCYC184, pACYC177, and pBR322. The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes. The repressor of the gut operon seemed to be encoded in the region downstream from the operon. The gene products of the gut operon were identified by using maxicells. The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.     

Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli

We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T. H. (1982) J. Biol. Chem. 257, 5125-5128). Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322. Restriction maps were prepared, and several DNA segments were subcloned into pBR322. Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment. The size of the melAB region was about 3,000 base pairs. Gene products were identified using maxicells harboring plasmids carrying the melibiose operon. The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.     

Physical and genetic structure of the IncN plasmid R15

Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.     

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.     

Physical and genetic structure of the maize genome reflects its complex evolutionary history

Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.     

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.     

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

Summary Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA ⁺ recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.