Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase

Abstract

L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspHI mutation at 2150. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t, to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2-to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspHI mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by to of sporulation but then showed a small increase, which was mirrored by changes in the level of ,-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulationspecific RNA polymerase tested yielded a -73 transcript in vitro.

Properties of purified sporlets produced by spoII mutants of Bacillus subtilis

“…The specific activities of aspartase and asparaginase were significantly higher in stage II sporlets than in t2 sporulating cells, a result indicating that most of these enzymes are produced in the forespore compartment of sporulating cells, as suggested previously (26). Enzymes of intermediary metabolism showed specific activities in stage II sporlets similar to or higher than those in t2 sporulating cells; these values were generally similar to those found previously with B. megaterium t2 sporulating cells or forespores isolated at t4 of sporulation (23).…”

Site‐specific mutagenesis of Escherichia coli asparaginase II

“…His1 97 is conserved in three high-affinity Alignments were made using the program MACAW [21]. EcoA2, E. coli asparaginase IT [22]; AgGA, Acinetabacter glutaminas$icans glutaminase-asparaginase [23] ; ErcA, asparaginase from Erwinia chrysanthemi [24]; BsuA, asparaginase from Bacillus subtilis [25]; YeaA2, Yeast asparaginase I1 [26]; EcoAl , E. coli asparaginase I [27]. Residues appearing in three or more of the sequences are shown in capitals, conservative replacements are indicated by italics.…”

Asparagine degradation in Rhizobium etli

“…Asparaginase activity has been studied in Escbericbia coli and other Gram-negative bacteria such as Salmonella enterica , Erwinia cbr_santbemi (Gilbert e t al., 1986) and Vibrio protetls (Sinha e t al., 1991), and in Gram-positive bacteria such as Bacillzrs subtilis (Sun & Setlow, 1991) and Stapbylococctls azlreas (Sobis & Mikucki, 1991 ;R6zalska & Mikucki, 1992). E. coli contains two Lasparaginase isozymes.…”

“…In some organisms, L-asparagine can be utilized as sole carbon and nitrogen source through the action of two enzymes (Sun & Setlow, 1991). The first enzyme, asparaginase (EC 3 .…”

“…However, the anaerobic regulation of this gene does not act via the FNR protein, contrasting with the case in E. coli (Jennings e t al., 1993). B. stlbtilis Lasparaginase and L-aspartase are encoded by the ans operon, which is subject to strong repression by a good nitrogen source, such as ammonium, and is also induced by asparagine and aspartate (Sun & Setlow, 1991;Atkinson & Fisher, 1991 ; Iijima e t a/., 1977). L-Aspartase activity has been studied in several bacteria, such as E. coli, B. stlbtilis (Sun & Setlow, 1991), Psetldomonas fltlorescens (Tokushige, 1985 ;Miyamoto & Katsuki, 1992) and Serratia naarcescens (Takagi & Kisumi, 1985).…”