Biomedical subjects

T M Henkin

Publications and source records attributed to T M Henkin.

At least 37 records · Page 2Linked to original sources

PubMed1994

Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA.

The Bacillus subtilis acsA (acetyl coenzyme A synthetase) and acuABC (acetoin utilization) genes were previously identified in the region downstream from the ccpA gene, which encodes a protein required for catabolite repression of the amyE (alpha-amylase) gene. The acsA and acuABC genes are divergently transcribed, with only 20 bp separating the -35 sequences of their promoters. Expression of these genes was maximal in stationary phase and was repressed by the addition of glucose to the growth medium. Two sites resembling amyO, the cis-acting regulatory target site for amyE, were identified in the acsA and acuABC promoter regions. Glucose repression of acsA and acuABC transcription was dependent on both CcpA and the amyO-like sequences.

Acetate-CoA Ligase↗

PubMed1994

Inducible amber suppressor for Bacillus subtilis.

An amber suppressor variant of Bacillus subtilis tyrosyl-tRNA was constructed and placed under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter. Addition of IPTG resulted in a 50-fold increase in the expression of an rpsD-lacZ fusion containing a UAG amber codon. This system permitted isolation of a conditional lethal mutant which required IPTG for growth.

Bacillus subtilis↗

PubMed1993

tRNA as a positive regulator of transcription antitermination in B. subtilis.

Most Bacillus tRNA synthetase genes are regulated by a common transcription antitermination mechanism but respond individually to limitation for the cognate amino acid. The mRNA leader regions of these genes exhibit extensive structural conservation, with a single codon specific for the appropriate amino acid at the identical position in each structure. Alteration of this sequence in the tyrS gene from UAC (tyrosine) to UUC (phenylalanine) resulted in loss of induction by tyrosine limitation and a switch to induction by phenylalanine limitation. Insertion of an extra base immediately upstream of the codon did not alter regulation, indicating a nontranslational mechanism. A nonsense codon resulted in an uninducible phenotype that was suppressible in a lysyl-tRNA nonsense suppressor mutant, indicating that tRNA acts as an effector.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis.

The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose. The region of the B. subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present. Two open reading frames that exhibited sequence similarity to the Escherichia coli and B. subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility. Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region. The acsA gene encodes acetyl-CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation. Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol. The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity. Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA-acuABC promoter regions.

Acetate-CoA Ligase↗

PubMed1993

Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E. coli gene closely related to ackA. Insertional inactivation of this region of the B. subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts. The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees. The transcriptional start site for B. subtilis ackA was located 90 bp upstream from the start of the coding region, and expression was increased by growth in the presence of excess glucose. Growth of the AckA- mutant was inhibited by glucose, suggesting that acetate kinase is important for excretion of excess carbohydrate. The stimulation of ackA expression by glucose was blocked in a CcpA- mutant, indicating that CcpA, which is required for glucose repression of certain carbon source utilization genes, including amyE, may also be involved in activation of carbon excretion pathways. Two sequences resembling the amyO operator site were identified upstream of the ackA promoter; removal of this region resulted in loss of glucose activation of ackA expression.

Acetate Kinase↗

PubMed1992

Characterization of the Bacillus subtilis rpsD regulatory target site.

The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation. Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene. A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested. These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point. Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B. subtilis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation. The rpsD leader region was isolated from several Bacillus species. Members of the B. subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B. subtilis, despite considerable primary sequence variation; the B. brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacillus species. Very little similarity to the Escherichia coli alpha operon S4 target site was found at either the primary-sequence or the secondary-structure level. Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains. The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacillus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.

Bacillus subtilis↗

PubMed1992

Analysis of the Bacillus subtilis tyrS gene: conservation of a regulatory sequence in multiple tRNA synthetase genes.

The Bacillus subtilis tyrS gene, which encodes tyrosyl-tRNA synthetase (TyrTS), was isolated, and its nucleotide sequence was determined. The cloned gene was shown to complement an Escherichia coli tyrS (Ts) mutant. The predicted amino acid sequence exhibited 70% identity to that of Bacillus stearothermophilus TyrTS and 55% identity to that of E. coli TyrTS, while identity to a second cryptic B. subtilis TyrTS gene, designated tyrZ, was only 27%. Primer extension analysis indicated that tyrS transcription initiated at a vegetative promoter sequence located 300 nucleotides upstream of the AUG start codon. The mRNA leader region was found to contain an inverted repeat sequence resembling a transcriptional terminator. Expression of a transcriptional tyrS-lacZ fusion was found to be induced by starvation for tyrosine in a tyrosine auxotroph (tyrA1). Transcription initiation was unaffected by tyrosine starvation. Deletion of the terminator region in a tyrS-lacZ fusion resulted in high-level constitutive expression. Immediately preceding the putative terminator was sequence element found to be conserved in the upstream region of a number of Bacillus tRNA synthetase genes as well as in the ilv-leu biosynthetic operon; mutation of this element in tyrS resulted in low-level uninducible expression. The conservation of this sequence element suggests that aminoacyl-tRNA synthetase genes and the ilv-leu operon may be regulated by a common mechanism in Bacillus spp.

Amino Acid Sequence↗

PubMed1991

Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors.

Expression of the alpha-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose-mediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263 degrees on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.

Acetoin↗

PubMed1991

The rpsD gene, encoding ribosomal protein S4, is autogenously regulated in Bacillus subtilis.

Although the mechanisms for regulation of ribosomal protein gene expression have been established for gram-negative bacteria such as Escherichia coli, the regulation of these genes in gram-positive bacteria such as Bacillus subtilis has not yet been characterized. In this study, the B. subtilis rpsD gene, encoding ribosomal protein S4, was found to be subject to autogenous control. In E. coli, rpsD is located in the alpha operon, and S4 acts as the translational regulator for alpha operon expression, binding to a target site in the alpha operon mRNA. The target site for repression of B. subtilis rpsD by protein S4 was localized by deletion and oligonucleotide-directed mutagenesis to the leader region of the monocistronic rpsD gene. The B. subtilis rpsD leader exhibits little sequence homology to the E. coli alpha operon leader but may be able to form a pseudoknotlike structure similar to that found in E. coli.

Amino Acid Sequence↗

PubMed1990

Cloning and analysis of the Bacillus subtilis rpsD gene, encoding ribosomal protein S4.

The rpsD gene, encoding ribosomal protein S4, was isolated from Bacillus subtilis by hybridization with oligonucleotide probes derived from the S4 amino-terminal protein sequence. Sequence analysis of the cloned DNA indicated that rpsD is likely to be monocistronic, in contrast to Escherichia coli rpsD, which is located in the alpha operon and is the translational regulator for alpha operon ribosomal protein gene expression in E. coli. The cloned gene was shown to map at position 263 degrees on the B. subtilis chromosome, at the position to which mutations conferring alterations in the electrophoretic mobility of protein S4 were localized. A promoter was identified upstream of the rpsD coding sequence; initiation of transcription at this promoter would result in a transcript containing a leader region 180 bases in length. Immediately downstream of the rpsD coding region were two sequences resembling transcriptional terminators. An open reading frame homologous to tyrosyl-tRNA synthetase (tyrS) genes was identified downstream of rpsD but in the opposite orientation. The leader region of rpsD mRNA is predicted to have extensive secondary structure, resembling a region of B. subtilis 16S rRNA where S4 is likely to bind; similar mRNA features have been found to be important in ribosomal gene regulation in E. coli. These results provide the first steps toward analysis of the regulation of rpsD gene expression in B. subtilis.

Amino Acid Sequence↗

PubMed1990

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.

Amino Acid Sequence↗

PubMed1989

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.

Amino Acid Sequence↗

PubMed1987

Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence.

The amyR1 locus controls the regulated transcription of amyE, the structural gene encoding alpha-amylase in Bacillus subtilis. Transcription of amyE is activated in early stationary phase cells, and can be repressed by rapidly metabolized carbon sources such as glucose. Transcription of amyE initiates in vitro from a promoter recognized by the major vegetative form of RNA polymerase, E sigma 43. S1 nuclease mapping of in-vivo amylase transcripts suggests that this promoter is also used in vivo. Two independently isolated cis-acting mutations, gra-5 and gra-10, which abolish glucose-mediated repression of amylase synthesis without altering temporal activation, were determined by DNA sequencing to result from a G.C to A.T transition at a position located five base-pairs downstream from the start site of transcription. While this is the first example of a site involved in catabolite repression of gene expression in a Gram-positive micro-organism, the region surrounding the gra mutations shows considerable homology to certain cis-acting regulatory loci in Escherichia coli, suggesting that such sequences have been evolutionarily conserved.

Amino Acid Sequence↗

PubMed1987

Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis.

The Escherichia coli lacUV5 promoter is used inefficiently by the major vegetative form of Bacillus subtilis RNA polymerase, despite very close adherence in the -35 and -10 regions to consensus sequences for promoters recognized by this enzyme. To select derivatives of this promoter with increased activity in B. subtilis, the lacUV5 promoter was fused to a promoter-less chloramphenicol acetyltransferase gene and mutagenized by passage through an E. coli mutD5 mutator strain. Derivatives that conferred resistance to chloramphenicol in B. subtilis were isolated. Twenty-three independent isolates each contained single mutations in the 207 bp lac fragment. These mutations, which were in ten different positions, fell in two clusters. One set of mutations, located between positions -18 and -14, resulted in greater homology to a consensus sequence previously noted for this region in B. subtilis vegetative promoters. The remaining mutations were located near the transcription initiation site. The effects of these mutations and additional mutations constructed by oligonucleotide-directed mutagenesis on expression in B. subtilis and E. coli was determined by measurements of chloramphenicol acetyltransferase activities directed by these promoters. While most mutations had little effect on expression in E. coli, the increase in activity in B. subtilis was as much as 28-fold.

Acetyltransferases↗

PubMed1985

Induction of the chloramphenicol acetyltransferase gene cat-86 through the action of the ribosomal antibiotic amicetin: involvement of a Bacillus subtilis ribosomal component in cat induction.

The plasmid gene cat-86 and the cat gene resident on pC194 each encode chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. Chloramphenicol induction has been proposed to result from chloramphenicol binding to ribosomes, which then permits the drug-modified ribosomes to perform events essential to induction. If this proposal were correct, B. subtilis mutants containing chloramphenicol-insensitive ribosomes should not permit chloramphenicol induction of either cat-86 or pC194 cat. However, we and others have been unable to isolate chloramphenicol-resistant ribosomal mutants of B. subtilis 168. We therefore developed a simple procedure for screening other antibiotics for the potential to induce cat-86 expression. One antibiotic, amicetin, was found to be an effective inducer of cat-86 but not of the cat gene on pC194. Amicetin and chloramphenicol each interact with the 50S ribosomal subunit, and the mechanism of cat-86 induction by both drugs may be similar. Amicetin-resistant mutants of B. subtilis were readily isolated, and in none of six mutants tested was cat-86 detectably inducible by amicetin, although the chloramphenicol-inducible phenotype was retained. The ami-1 mutation which is present in one of these amicetin-resistant mutants was mapped by PBS1 transduction to the "ribosomal gene cluster" adjacent to cysA. Additionally, ribosomes from cells harboring the ami-1 mutation contained an altered BL12a protein, as detected in two-dimensional polyacrylamide gel electrophoresis. Lastly, an in vitro protein-synthesizing system that uses ribosomes from an ami-1-containing cell line was more resistant to amicetin than a system that uses ribosomes from an amicetin-sensitive but otherwise isogenic strain. These results indicate that the host mutation, ami-1, which effectively abolished the inducibility of cat-86 by amicetin, altered a ribosomal component.

Acetyltransferases↗

PubMed1984

Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4.

A mutation causing an alteration in Bacillus subtilis ribosomal protein S4 was mapped by transformation and PBS-1 transduction to a site between aroG and argA, a region of the B. subtilis chromosome not previously demonstrated to contain ribosomal protein genes. The S4 mutation conferred a spore-plus phenotype in a streptomycin-resistant, spore-minus genetic background. The altered protein was detectable by polyacrylamide gel electrophoresis of ribosomal proteins of recombinants scored for the spore-plus phenotype in genetic crosses.

Bacillus subtilis↗

PubMed1984

Genetic analysis of a streptomycin-resistant oligosporogenous Bacillus subtilis mutant.

Strain SRB15T+, a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis, contains two mutations, fun and strR. These mutations were mapped by PBS-1 mediated transduction and by transformation to two different sites in the cysA-linked region of the B. subtilis chromosome. The fun mutation mapped very close to rpsLl, a classic strA mutation, whereas strR mapped to a site distal to rpsE. The effects of these mutations on growth, sporulation, and streptomycin resistance in vivo and in vitro were determined. The fun mutation gave a different phenotype than did the rpsLl mutation and caused altered migration of a ribosomal protein which was identified as S12, the protein encoded by rpsL. It therefore appears that fun is an allele of the rpsL gene.

Alleles↗

PubMed1984

New chloramphenicol resistance locus in Bacillus subtilis.

A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal. Its presence does not confer dependence on chloramphenicol. Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis. No chloramphenicol acetyltransferase activity could be detected.

Acetyltransferases↗