Hermine L. Bingham

Complete sequence of the Campylobacter jejuni glyA gene encoding serine hydroxymethyltransferase

The complete nucleotide sequence of the Campylobacter jejuni glyA gene was determined and the amino acid (aa) sequence of its product, serine hydroxymethyltransferase (SHMT), was deduced. The deduced polypeptide has 414 aa residues (Mr 45,758). The aa sequences of C. jejuni and Escherichia coli show 55.6% identity. Comparative analysis of the aa sequences of the SHMTs of E. coli and C. jejuni identified two new putative functional domains. The translational product of the C. jejuni glyA gene was identified using both minicell and maxicell systems and the transcription start point was mapped. The deduced transcription-regulatory signals, -10 and -35 sequences, show high homology to the corresponding consensus sequences for sigma 70 promoters in E. coli. The C. jejuni glyA promoter may be useful in the construction of shuttle vectors between E. coli and C. jejuni.

Cloning and sequence analysis of the flagellin gene ofCampylobacter jejuni TGH9011

Flagella are essential for motility and have been implicated to be one of the pathogenic determinants. The flagellum ofCampylobacter jejuni is a polymeric structure of a 62-kd protein. Using a high-affinity flagellin antibody to screen a lambda gt 11 phage genomic expression library ofC. jejuni strain TGH9011 (Serotype LIO36), a recombinant phage clone lambda gt 11RK that expresses theC. jejuni flagellin protein was isolated. The recombinant lambda gt 11 RK produced a 56-kd protein upon induction with isopropylthiogalactoside, which reacted specifically with anti-flagellin antibody. The flagellin gene was sequenced, and comparative analysis of the nucleotide and amino acid sequence identified a region of the flagellin that shows hypervariability among differentCampylobacter species and strains.

Cloning and expression of the Campylobacter jejuni glyA gene in Escherichia coli

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.

The multisite character of host-range mutations in bacteriophage λ

Mapping of the h and hh * host-range mutations in phage lambda by two-point crosses with reference J- point mutations, and with lambdagal deleted for part of J, locates these mutations in the promoter-distal portion of the J cistron. Analysis of phenotypic h+ recombinants, formed in crosses of the type h or hh* x J-, or h+ revertants of h, hh* , and Jdef mutants, indicates that such phenotypic h+ particles often retain cryptic h determinants. Similar determinants are also present in some common laboratory strains of lambda. These h+ recombinants and revertants carry a variety of different h markers, since recombination analysis allows several classes of particles carrying cryptic h markers to be distinguished. These genetic data suggest that the extended host-range phenotype in lambda is due to multiple rather than single, mutations in the distal region of gene J, although the number of sites involved and their arrangement remain uncertain. The genetic location of the h and hh * mutations is confirmed at the physical level by comparing the tryptic peptide maps of the J proteins purified from lysates of cells infected with different h+, h, hh*, Jam, and J434 phage and from purified lambdah+ virions. Examination of these peptide maps shows there are several methionine-containing peptides altered in the h and hh * maps. Some of these altered peptides are derived from the C-terminal 5-10% of the J polypeptide in the region of nonhomology between lambda and 434.

Genetic and physiological characterization of the J gene of bacteriophage lambda.

Abstract More than 20 temperature-sensitive and defective J mutants of λ have been analyzed in terms of the location of the mutations and their effect on the gene product. All of the defective sites and most of the temperature-sensitive sites are confined to a comparatively small region at the right-hand end of the J map. This is the region in which genetic determinants of the distinctive host-range and serological characteristics of phages λ and 434 are located. Although most of the λ defective mutants do not produce an active J protein, two mutants with closely linked mutations produce material that is serologically active and can be incorporated into intact particles. Leakiness of the temperature-sensitive J mutants is highly variable, in a way that suggests that the location of the codon change has a bearing on how readily polypeptides produced at high temperature undergo transition to a temperature-insensitive state.

Imbalance of purine nucleotides in alanosine-resistant baby hamster kidney cells

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK+ cells in medium containing alanosine, adenosine, and uridine (AAV medium). Twenty AAUr isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAUr cells were able to convert exogenous adenosine → inosine → hypoxanthine → IMP → AMPS → AMP. Moreover, these AAUr cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAUr cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK+ BHK cells were isolated.

Temperature sensitivity in Esherichia coli K12: Mutants unable to support normal growth of γ phage at high temperatures

Abstract Physiological analyses of seven temperature-sensitive mutants of Escherichia coli K12 suggest that the inability of two of the mutants to support normal λ reproduction at high temperature is associated with a defect in λ DNA synthesis. Failure of one of the mutants to support the growth of λ and two other phages is attributed to a temperature-sensitive block in protein synthesis. Abnormalities in λ development in the remaining mutants appear to reflect participation of different temperature-sensitive host components in processes occurring comparatively late in the phage growth cycle. The approximate map positions of mutations responsible for temperature sensitivity of four of the mutants have been determined.