Hideho Suzuki

New topoisomerase essential for chromosome segregation in E. coli

The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous to that of the A subunit of gyrase. We found another new gene coding for about 70 kd protein. The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit. Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE. Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers. A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage. It is suggested that the parC and parE genes code for the subunits of a new topoisomerase, named topo IV.

Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium

SummaryThrough genetic studies, the fliA gene product has been shown to regulate positively gene expression in late operons of the flagellar regulon in Salmonella typhimurium. In the present study, the fliA gene was cloned and sequenced. The fliA coding region consisted of 717 nucleotides beginning with the GTG initiation codon and the conserved sequence specific to promoters for flagellar operons was found to exist upstream of the coding region. The fliA gene product deduced from the nucleotide sequence was a protein with 239 amino acid residues and the calculated molecular mass was 27470 dalton. The deduced amino acid sequence was homologous with that of σ28, a flagellar specific sigma factor of Bacillus subtilis. The fliA gene product was identified as a protein of molecular mass 29 kDa in the in vitro transcription-translation system, while three proteins of 29 kDa, 31 kDa and 32 kDa were found in the products programmed by the fliA gene in minicells and in maxicells. The 29 kDa FHA protein was purified from the FliA overproducing strain which carried the ptac-fliA fusion. This protein activated the in vitro synthesis of flagellin, the fliC gene product. RNA polymerase containing the purified FliA protein was shown to transcribe the fliC gene. These results indicate that FliA protein functions as an alternative sigma factor specific for S. typhimurium flagellar operons.

On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3.

SummaryWe determined the nucleotide sequence of a DNA fragment containing the ftsI gene coding for the penicillin-binding protein 3 (PBP-3), an indispensable enzyme for cell division of Escherichia coli. The entire ftsI gene was within the 2.8 kilobase PvuII fragment derived from the chromosomal segment on pLC26-6 (Nishimura et al. 1977). The coding region for PBP-3 was identified by comparison with the N-terminal amino acid sequence of in vitro synthesized PBP-3. The structural gene for ftsI consisted of 1,764 base-pairs coding for a 588 amino acid residuepolypeptide with a molecular weight of 63,850. PBP-3 synthesized in vitro showed a lower mobility in SDS-gel electrophoresis than that of the authentic PBP-3, suggesting that the primary translation product of the ftsI gene may be processed to yield mature PBP-3.

Dispensability of either penicillin-binding protein-1a or -1b involved in the essential process for cell elongation in Escherichia coli.

SummaryA strain of Escherichia coli lacking the entire ponB gene and a strain lacking the proximal part of the ponA gene were constructed by substitution with a drug resistance gene. These strains lost either penicillin-binding protein(PBP) -1b or -1a totally and their growth was apparently normal at 30°C and 42°C except that growth of the ponB deletion strain was poor on a nutrient agar plate containing no NaCl at 30°C as well as at 42°C. Transductional experiments to introduce the ponB deletion into the ponA deletion strain, and vice versa, showed that the ponA ponB double deletion was lethal unless the deletion was functionally compensated, e.g., by the presence of a plasmid carrying either gene. Thus, either PBP-1b (ponB) or PBP-1a (ponA), but not both, is dispensable for cell viability, at least under ordinary culture conditions. Transductional experiments also suggested that the γ component of PBP-1b or the PBP-1b lacking the C-terminal portion encoded in the distal region to the SphI site on the ponB was sufficient for supporting growth of the E. coli cell.

The role of cAMP in flagellation of Salmonella typhimurium.

SummaryA mutational alteration either in adenylate cyclase (cya-) or in cyclic-3′5′-AMP (cAMP) receptor protein (crp-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya- mutation, was identified among the revertants of cya-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.

A novel glycan polymerase that synthesizes uncross-linked peptidoglycan in Escherichia coli.

A simple and efficient procedure to assay peptidoglycan synthesis in vitro was established. By this procedure, a novel activity for glycan polymerization in Escherichia coli was found in the fraction containing no detectable penicillin‐binding protein (PBP). This polymerase activity was relatively insensitive to moenomycin, showed requirement for Ca2+ or Mn2+ but not for Mg2+, and led to production of uncross‐linked glycan chains. These properties distinguished the glycan polymerase from the activities shown by the fractions containing PBPs. The glycan polymerase catalyzing polymerization of glycan units from lipid intermediates was purified and identified as a protein of 34 kDa.

Escherichia coli parA is an allele of the gyrB gene

SummaryA thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4′,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.

Overlapping of the coding regions for α and γ components of penicillin-binding protein 1 b in Escherichia coli

SummaryThe mode of biosynthesis of penicillin-binding protein(PBP)-1 b in Escherichia coli was investigated by use of the plasmid carrying the ponB(PBP-1 b) gene region. Analyses of the products synthesized in minicells and in vitro showed that PBP-1 b was synthesized as two molecular species corresponding to the α and γ components of PBP-1 b. The coding regions for the α and γ components were located within the ca. 3.7 kb MluI-HincII fragment and transcribed in the direction from the HincII to the MluI site. The capacity for producing the α component was abolished by a deletion extending to the MluI site ca. 0.7 kb inward from the HincII end of the ca. 3.7 kb fragment; the remaining 3.0 kb region with the MluI site at both ends directed the production of the γ component alone. The production of the γ component was enough to correct all the known defects caused by a ponB mutation. In addition to these results, the analyses for cross-reacting materials produced in correspondence to the various deletions indicated that the coding regions for the α and γ components overlapped and that the N-terminal portion was responsible for the difference between the two components. The distal region about 0.7 kb long inward from the MluI end of the MluI-HincII fragment was dispensable for producing the functional PBP-1 b, although the PBP-1 b produced was curtailed. By a larger distal deletion reaching almost to the middle of the MluI-HincII fragment, the polypeptide produced for PBP-1 b lost the ability to bind penicillin and still retained a low but significant activity for glycan synthesis. We suggest, therefore, that the polypeptide portion required for transglycosylase activity resides on the N-terminal half of PBP-1 b, followed by the middle portion necessary for penicillin-binding and the C-terminal part dispensable for the function of PBP-1 b.

Absence of messenger ribonucleic acid specific for flagellin in non-flagellate mutants of Salmonella.

Abstract The flagellin synthesizing activity of RNAs prepared from the fla mutants of Salmonella was examined using the Escherichia coli cell-free system for protein synthesis. The fla complementation groups tested covered the following: fla-A, -B, -C, -D, -E and -F in Salmonella typhimurium and fla-L, -N, -P, -Q and -R in S. abortus-equi . None of the RNAs were found to stimulate in vitro synthesis of flagellin, although stimulation of amino acid incorporation into general protein was high enough. Complementation at a translational level was not observed in any of the following combinations of the RNAs prepared from the fla mutants: A-F, B-C, B-E, C-D, E-F, F-L and N-P . A mutation in one of the fla genes tested resulted in failure to produce flagellin mRNA in both flagellar phrases. The products of fla + were assumed to participate in the transcription of the structural gene for flagellin.

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single‐celled Protonemata of the Fern Adiantum Capillus‐veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single‐celled protonemata of the fern Adiantum capillus‐veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with 3H‐thymidine during spore germination so that the amount of 3H incorporated into the TCA‐insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of 14C‐amino acids into TCA‐insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with 35S‐methionine at various times after the transfer from red light condition (G0) to darkness (G1 to S) were analyzed by two‐dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G1, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G1 and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.