Genetic Analysis of a Chromosomal Region Containing Genes Required for Assimilation of Allantoin Nitrogen and Linked Glyoxylate Metabolism in Escherichia coli

Abstract

ABSTRACT Growth experiments with Escherichia coli have shown that this organism is able to use allantoin as a sole nitrogen source but not as a sole carbon source. Nitrogen assimilation from this compound was possible only under anaerobic conditions, in which all the enzyme activities involved in allantoin metabolism were detected. Of the nine genes encoding proteins required for allantoin degradation, only the one encoding glyoxylate carboligase ( gcl ), the first enzyme of the pathway leading to glycerate, had been identified and mapped at centisome 12 on the chromosome map. Phenotypic complementation of mutations in the other two genes of the glycerate pathway, encoding tartronic semialdehyde reductase ( glxR ) and glycerate kinase ( glxK ), allowed us to clone and map them closely linked to gcl . Complete sequencing of a 15.8-kb fragment encompassing these genes defined a regulon with 12 open reading frames (ORFs). Due to the high similarity of the products of two of these ORFs with yeast allantoinase and yeast allantoate amidohydrolase, a systematic analysis of the gene cluster was undertaken to identify genes involved in allantoin utilization. A BLASTP search predicted four of the genes that we sequenced to encode allantoinase ( allB ), allantoate amidohydrolase ( allC ), ureidoglycolate hydrolase ( allA ), and ureidoglycolate dehydrogenase ( allD ). The products of these genes were overexpressed and shown to have the predicted corresponding enzyme activities. Transcriptional fusions to lacZ permitted the identification of three functional promoters corresponding to three transcriptional units for the structural genes and another promoter for the regulatory gene allR . Deletion of this regulatory gene led to constitutive expression of the regulon, indicating a negatively acting function.