Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

Nucleotide sequence of the Vibrio alginolyticus calcium-dependent, detergent-resistant alkaline serine exoprotease A

The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.     

The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology-facilitated illegitimate recombination during transformation of Acinetobacter baylyi

Homology-facilitated illegitimate recombination (HFIR) promotes genomic integration of foreign DNA with a single segment homologous to the recipient genome by homologous recombination in the segment accompanied by illegitimate fusion of the heterologous sequence. During natural transformation of Acinetobacter baylyi HFIR occurs at about 0.01% of the frequency of fully homologous recombination. The role of the 5' single-strand-specific exonuclease RecJ in HFIR was investigated. Deletion of recJ increased HFIR frequency about 20-fold compared with wild type while homologous recombination was not affected. Illegitimate fusion sites were predominantly located within 360 nucleotides away from the homology whereas in wild type most fusion sites were distal (500-2500 nucleotides away). RecJ overproduction reduced the HFIR frequency to half compared with wild type, and transformants with short foreign DNA segments were diminished, leading to on average 866 foreign nucleotides integrated per event (682 in wild type, 115 in recJ). In recJ always the 3' ends of donor DNA were integrated at the homology whereas in wild type these were 3' or 5'. RecJ apparently suppresses HFIR by degrading 5' non-homologous DNA tails at the post-synaptic stage. We propose that the RecJ activity level controls the HFIR frequency during transformation and the amount of foreign DNA integrated per event.     

Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot.

Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage. Such recombination is rare, but it is greatly enhanced by UV irradiation. We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold. In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant. Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot. These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot. Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences. We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot.     

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy. The deduced amino acid sequence suggests a protein consisting of 338 residues with an Mr of 37,500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.     

Nucleotide sequence of the gene for Escherichia coli ribosomal protein S15 (rpsO)

Summary The nucleotide sequence of the ribosomal protein gene rpsO (S15) and its flanking region were determined. The amino acid sequence of S15 protein deduced from the nucleotide sequence is in good agreement with the published amino acid sequence with one exception. The nucleotide sequence shows two probable promoter sites about 100 nucleotides upstream from the initiation codon (AUG) of rpsO. Inspection of the sequence also revealed structural homology between the distal part of rpsO and the reported S15 binding region in 16S rRNA.