Mannopinic acid and agropinic acid catabolism region of the octopine-type Ti plasmid pTi15955

Octopine-type Ti plasmids such as pTi15955, pTiA6 and pTiR10 direct the catabolism of at least eight compounds called opines that are released from crown gall tumours. Four of these compounds are denoted mannityl opines, each of which possesses a D-mannityl substituent on the nitrogen atom of either glutamate or glutamine. We have analysed a 20 kb region of the Ti plasmid pTi15955 that is required for the catabolism of two such opines, mannopinic acid and agropinic acid. A total of 12 genes in four operons were identified by DNA sequence analysis. Transposons Tn5lacZ and MudK were used to mutagenize these genes and to create aga-lacZ and moa-lacZ translational fusions. The expression of all fusions was induced by agropinic acid and by mannopinic acid. One of these four operons encodes an agropinic acid permease, whereas a second one encodes a mannopinic acid permease. A third operon contains three genes encoding probable catabolic enzymes, two of which (AgaF and AgaG) are thought to convert agropinic acid to mannopinic acid, while the third (AgaE) probably converts mannopinic acid to mannose and glutamate. AgaE resembles a bacterial amino acid deaminase, whereas AgaF and AgaG resemble two bacterial proteins that together catabolize substituted hydantoins, whose chemical structure resembles that of agropinic acid. The remaining operon encoded the MoaR protein, a negative regulator of itself and of the other three operons.     

Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA. A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region. A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation. Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.     

A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.