Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate.

The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

Newly identified genes involved in the signal transduction of Escherichia coli K-12.

We cloned and sequenced two Escherichia coli genes which are members of a family of an environmentally responsive two-component system. The nucleotide (nt) and deduced amino-acid sequences of these two genes were found to be homologous to those of the Bordetella pertussis bvgA and bvgS genes. They were mapped at 51 min (clones 6B9 to 7G9 of the Kohara miniset library of the E. coli chromosome). Both proteins, deduced from their nt sequences, were identified in the coupled in vitro transcription-translation system; their molecular masses were consistent with BvgA and BvgS (23 and 135 kDa, respectively). Furthermore, when these genes were expressed on a multicopy plasmid in an envZ deletion strain, ompC expression was induced. This expression was found to be regulated by low temperature, MgSO4 and nicotinic acid, factors known to control the virulence of B. pertussis via BvgA and BvgS. These results indicate that the newly cloned genes were structurally and functionally similar to bvgA and bvgS, and we designated these genes evgA and evgS.

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli.

The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene.

Functional analysis of the fic gene involved in regulation of cell division.

We have shown that cAMP may be a regulation factor in cell division of Escherichia coli (Utsumi et al., 1982, 1989). The fic (filamentation induced by cAMP) gene of this system found previously (Utsumi, et al., 1982) was analysed in this study. The open reading frame of the fic gene coded for 200 amino acids. The pabA (p-aminobenzoate synthase) gene was found downstream from the fic gene. The distance between the end of fic and the start of pabA was 31 base pairs. To deduce the function of Fic protein, the fic gene was destroyed by the kanamycin-resistant (Kmr) gene and the fic gene was shown to be essential for growth of E. coli. Such mutants required PAB (p-aminobenzoate) or folate for growth. These data suggested that the Fic protein is involved in the synthesis of PAB or folate and the fic gene could be part of a pab operon. In cells starved of them, cell division was inhibited. Addition of folate also repressed the filamentation induced by cAMP at 43 degrees C in the fic-1 mutant. These results would indicate that Fic protein and cAMP are involved in a new regulatory mechanism of cell division via folate metabolism. Furthermore, it is also shown that cell division could be controlled by coordination of cAMP, Fic and Fts proteins.

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124.

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).

Stationary phase-specific expression of the fic gene in Escherichia coli K-12 is controlled by the rpoS gene product (sigma 38).

The fic gene, near pabA located at 75 min of the Escherichia coli chromosome, was previously identified as the regulatory factor of cell division. In this paper we have examined how fic gene expression is controlled during the growth cycle using a fic-lacZ protein fusion plasmid (pFL1). Its expression was induced at stationary phase while it was nearly abolished in rpoSmutants. Using a RNase protection assay, fic transcript at stationary phase was detected in rpoS+ strains, but not in the rpoS mutants. Furthermore, primer extension analysis indicated that the fic transcript controlled by RpoS initiates at a G located 185 bp upstream from ATG of the fic coding region. Compared with the sigma 70 recognition sequence, the -10 region of fic promoter resembled the Pribnow box, but no homologous sequence was observed at the -35 region. These results were consistent with the characteristic sequence profile of fic promoter recognized specifically by RpoS in vitro, which is the only example of the type III promoter so far detected in vitro and in vivo.

Negative regulation of the bolA1p of Escherichia coli K-12 by the transcription factor OmpR for osmolarity response genes.

To test whether OmpR is involved in regulation of the bolA1p, we investigated possible effects of ompR mutation on transcription from bolA1p. In vivo, bolA1p was found to be repressed by OmpR. Furthermore in vitro, the phospho-OmpR was found to bind to the OmpR binding region of bolA1p and repress the transcription by Esigma(S) or Esigma(D). These results suggest that the phosphorylated form of OmpR is a negative regulator for the transcription of the bolA1p promoter.

Genotyping for RhC/c and RhE/e by PCR using allele-specific oligonucleotide primers.

The Rh blood group system has five major antigens D, C/c, and E/e. These antigens are encoded in RHD and RHCE genes. In this report, we describe a systemic method for RhC/c and RhE/e genotyping by PCR using allele-specific oligonucleotide primers (ASO-PCR). The ASO-PCR was carried out to determine the RhC/c and RhE/e genotypes in DNA samples from 513 Japanese donors. Genotypes of RhC, RhE, and Rhe were in full concordance with serological phenotypes in 511 donors. However, in two cases with the phenotype of ccdee, the C-specific ASO-PCR product was also detected in addition to the c-specific one. This method is simple and quite useful for the RhC/c and RhE/c genotyping, although further investigation on the 2 exceptional ccdee cases is needed.

Molecular characterization of the PhoP-PhoQ two-component system in Escherichia coli K-12: identification of extracellular Mg2+-responsive promoters.

We identified Mg2+-responsive promoters of the phoPQ, mgtA, and mgrB genes of Escherichia coli K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.

Intranuclear microinjection for transformation of tomato callus cells.

An efficient method, called the culture plate method, was devised for microinjection of foreign materials into nuclei of tomato callus cells. The culture plate method, used in this study, is advantageous because cells suitable for microinjection can be selected microscopically and the injected cells subsequently cultured in the same plate. With this microinjection system, some foreign materials were injected into nuclei of callus cells without causing detrimental effects. Kanamycin-resistant callus clones were obtained 1 month after injection from single cells whose nuclei were microinjected with a NPT II DNA fragment of the pE2KX plasmid.

Control mechanism of the Escherichia coli K-12 cell cycle is triggered by the cyclic AMP-cyclic AMP receptor protein complex.

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.

Binding of the protein from Thermus aquaticus ISLtaq1 to its inverted repeat in vitro.

We have isolated from Thermus aquaticus an insertion-sequence-like genetic element (ISLtaq1) that induces thermotolerance and has a high sequence similarity to IS150 belonging to the IS3 family. An open reading frame on ISLtaq1, termed ORF1, encodes the ORF1 protein, which carries a DNA-binding motif. In this study, we found an imperfect inverted repeat in ISLtaq1. We next overproduced and purified a His-tagged ORF1 protein. Gel retardation analysis demonstrated that this protein specifically bound to an DNA fragment containing the inverted repeat in ISLtaq1. These results suggest that ISLtaq1 and the ORF1 protein are an insertion sequence and part of the transposase encoded by ISLtaq1, respectively.

Antibacterial agents that inhibit histidine protein kinase YycG of Bacillus subtilis.

We demonstrated in vitro that YycG-YycF of Bacillus subtilis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner. Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.

Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108.

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.

Transcription of emrKY is regulated by the EvgA-EvgS two-component system in Escherichia coli K-12.

Spontaneous mutations have been isolated in Escherichia coli that result in the constitutive expression of an emrKY promoter. These mutations were found to be single-nucleotide substitutions within the linker region of the sensor protein EvgS, which is part of a two-component regulatory system along with EvgA. In the linker mutants (evgSI and evgS4), emrKY expression became constitutive and MIC against sodium deoxycholate was 20 mg/ml, eight-fold higher than in the wild type. Furthermore, the start site of transcription from the promoter of emrKY was identified; EvgA was shown to bind at the -52 to -84 region by the footprinting experiment.

Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids.

We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription.

Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.

The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin.

Ca2(+)-enhanced phosphorylation of a chimeric protein kinase involved with bacterial signal transduction.

The Tar-EnvZ hybrid molecule (Taz1) is an inner membrane transducer that activates OmpR, a transcriptional activator for porin gene expression (ompC), in response to an aspartic acid signal. Signal transduction by Taz1 most likely involves a phosphorylated Taz1 intermediate that donates its phosphate to OmpR. Phosphorylated OmpR has already been implicated in transcriptional activation of porin genes. Using a cell-free system containing Taz1-enriched membrane fractions, we have examined the phosphorylation properties of Taz1 and the stimulatory effects of divalent and monovalent ions. Highest activation of Taz1 phosphorylation was observed with CaCl2, and its stimulation could be observed with as low as 60 microM of CaCl2. Phosphorylated Taz1 could readily donate its phosphate group to OmpR in the presence of calcium. CaCl2 was also able to enhance phosphorylation of intact membrane-bound EnvZ and a cytoplasmic fragment of EnvZ lacking the receptor and transmembrane domains. These results indicate that the site for CaCl2 stimulation is within the cytoplasmic region of EnvZ and probably involves an enhanced rate of EnvZ phosphorylation.

Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant.

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.