Nucleotide sequence of gene linA encoding resistance to lincosamides in Staphylococcus haemolyticus

We have determined the nucleotide sequence of gene linA in plasmid pIP855, which confers resistance to lincomycin by inactivating it in Staphylococcus haemolyticus. The gene was defined by start and stop codons and an open reading frame of 483 bp corresponding to a product with an Mr of 19020. The apparent size of the resistance-conferring protein was 21 kDa, when a minicell extract was electrophoresed in the sodium dodecyl sulfate-polyacrylamide gels. Full expression of linA was obtained both in Escherichia coli and Bacillus subtilis.     

Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? A review

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeats present in up to 1000 copies on the Escherichia coli chromosome. We have shown both in vivo and in vitro that REP sequences can stabilize upstream mRNA by blocking the processive action of 3'----5' exonucleases. In a number of operons, mRNA stabilization by REP sequences plays an important role in the control of gene expression. Furthermore, differential mRNA stability mediated by the REP sequences can be responsible for differential gene expression within polycistronic operons. Despite the key role of REP sequences in mRNA stability and gene expression in a number of operons, several lines of evidence suggest that this is unlikely to be the primary reason for the exceptionally high degree of sequence conservation between REP sequences. Other possible functions for REP sequences are discussed. We propose that REP sequences may be a prokaryotic equivalent of 'selfish DNA' and that gene conversion may play a role in the evolution and maintenance of REP sequences.     

Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules.     

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.     

Structural and biochemical characterization of the Escherichia coli argE gene product.

The DNA sequence of a 2,100-bp region containing the argE gene from Escherichia coli has been determined. The nucleotide sequence of the ppc-argE intergenic region was also solved and shown to contain six tandemly repeated REP sequences. Moreover, the oxyR gene has been mapped on the E. coli chromosome and shown to flank the arg operon. The codon responsible for the translation start of argE was determined by using site-directed mutants. This gene spans 1,400 bp and encodes a 42,350-Da polypeptide. The argE3 allele and a widely used argE amber gene have also been cloned and sequenced. N-Acetylornithinase, the argE product, has been overproduced and purified to homogeneity. Its main biochemical and catalytic properties are described. Moreover, we demonstrate that the protein is composed of two identical subunits. Finally, the amino acid sequence of N-acetylornithinase is shown to display a high degree of identity with those of the succinyldiaminopimelate desuccinylase from E. coli and carboxypeptidase G2 from a Pseudomonas sp. It is proposed that this carboxypeptidase might be responsible for the acetylornithinase-related activity found in the Pseudomonas sp.     

The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.     

Nucleotide sequence of the amicyanin gene from Methylobacterium extorquens AM1.

The gene for amicyanin from the methylotrophic bacterium, Methylobacterium extorquens AM1 was identified. It encodes a protein consisting of 119 amino acids with a molecular weight of 12,609 kDa. The amino acid sequence shows the presence of a typical leader sequence and signal peptidase recognition site. Two putative hairpin structures were found, one located directly behind the amicyanin gene and another located 50 bp upstream. The same sequence AAAATCCC was found near the start codons for the small subunit of methylamine dehydrogenase and amicyanin, but its significance is not known.     

Sequences of the Escherichia coli photolyase gene and protein

We have determined the nucleotide sequence of a 2039-base pair segment of Escherichia coli chromosomal DNA containing the phr gene, which encodes deoxyribopyrimidine photolyase. The coding region of phr is 1416 base pairs and is preceded by regions homologous to consensus sequences for E. coli promoters and ribosome binding sites. The phr gene is preceeded by an open reading frame of 169 codons (orf169) which is transcribed in the same direction. The proximity of orf169 to phr suggests that both are members of a single operon containing one or more internal promoters allowing differential expression of phr. An unusually large number of rare or infrequently used codons are utilized in phr, which may contribute to the low copy number of photolyase. The sequence at the NH2 and COOH termini and the overall amino acid composition of mature photolyase, determined using purified protein, agrees with predictions based upon the nucleotide sequence. Photolyase consists of 471 amino acids and has a calculated molecular weight of 53,994.     

Characterization of the rfc region of Shigella flexneri.

The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an O-antigen-specific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one O-antigen repeat unit. Thus, the mutants are deficient in production of the O-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamycin resistance cartridge into a SalI site in this region inactivated the rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a protein with a predicted molecular mass of 43.7 kDa. The predicted protein is highly hydrophobic and showed little sequence homology with any other protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enterica (muenchen) revealed that the profiles were similar and that the proteins have 12 or more potential membrane-spanning segments. A comparison of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage of G + C content and have similar codon usage, and all have a high percentage of rare codons. An attempt to identify the S. flexneri Rfc protein was unsuccessful, although proteins encoded upstream and downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G + C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.