Nucleotide sequences of serine tRNAs from Bacillus subtilis.

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).     

Nucleotide sequence of the trpC-trpB intercistronic region from Salmonella typhimurium.

We have sequenced a DNA segment that contains the Salmonella typhimurium trpC-trpB junction. A series of 11 amino acids predicted from the sequence are identical to the amino-terminal amino acid sequence of Escherichia coli tryptophan synthetase β (Crawford et al., 1979). Carboxypeptidase A digestion of phosphoribosyl-anthranilate isomerase-indoleglycerolphosphate synthetase identified its carboxy-terminal amino acids allowing us to specify the end of trpC. Nine nucleotides separate the terminator codon of trpC from the initiator codon of trpB. The messenger RNA around the trpB initiation site, as well as around many other prokaryotic ribosome binding sites, has the potential to form stable stem and loop structures. These secondary structures share the property of having most, if not all, of the sequences complementary to the 3′ end of 16 S ribosomal RNA, as well as the initiator codon, included in single-stranded regions.     

Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences.

The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.     

Cluster of genes controlling proline degradation in Salmonella typhimurium.

A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).     

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Regulation of the major proline permease gene of Salmonella typhimurium.

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.     

Nucleotide sequence analysis of purH and purD genes from Salmonella typhimurium

The purH and purD genes coding for the 5'-phosphoribosyl 5-amino-imidazole-4-carboxamide (AICAR) transformylase and 5'-phosphoribosyl-glycinamide (GAR) synthetase, respectively, were identified on a 4.8 kb Eco RI fragment of chromosomal DNA from Salmonella typhimurium. Nucleotide sequence analysis of the cloned fragment revealed the presence of two large open reading frames (O.R.F.), which were separated by 11 base pairs (bp). Substantial DNA and amino acid sequence homology was noted between the purH and purD genes of S. typhimurium and Escherichia coli. Expression of the Salmonella purD gene in a T7 polymerase/promoter system revealed the presence of a 49 kDa protein band by SDS-PAGE and subsequent autoradiography. The purH gene of Salmonella was not expressed since the 5' end of this gene was not cloned.