Viruses, variants and vaccines.

The current SARS-CoV-2 pandemic has brought a number of major global clinical, sociological and economic issues into sharp focus. We address some of these issues, focusing on short-term factors such as virus mutations and vaccine efficacy, and also considering the longer-term implications of the current pandemic. We discuss societal responses to the presence of a pathogen that will probably remain in circulation for decades or longer, and to future new emergent viruses.

Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S.

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding D-amino acids or L-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase ( N-carbamoylase). Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and L-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and beta-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not beta-ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or beta-ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3S cells.

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens.

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.

The UGA4 UASNTR site required for GLN3-dependent transcriptional activation also mediates DAL80-responsive regulation and DAL80 protein binding in Saccharomyces cerevisiae.

Expression of the nitrogen catabolic genes in Saccharomyces cerevisiae, including those of the gamma-aminobutyric acid (UGA) and allantoin (DAL) pathways, is regulated positively by the GLN3 protein and negatively by the DAL80 protein. The deduced sequences of the DAL80 and GLN3 proteins contain a zinc finger motif homologous to those shown to bind GATA sequences. In addition, DAL80 protein has been directly shown to bind to a pair of GATA-containing sequences (URSGATA) in vitro, and a pair of GATA-containing sequences (UASNTR) is required for GLN3-dependent transcriptional activation in a heterologous expression vector. We demonstrate here that the GATA-containing sites upstream of UGA4 required for optimal GLN3-dependent transcriptional activation also mediate DAL80 protein binding in vitro and DAL80-responsive regulation in vivo.

The DAL82 protein of Saccharomyces cerevisiae binds to the DAL upstream induction sequence (UIS).

Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in S. cerevisiae is induced by allophanate, the last intermediate in the allantoin catabolic pathway. Analysis of the DAL7 promoter identified a dodecanucleotide, the DAL7 UIS, which was required for inducer-responsiveness. Operation of the DAL7 UIS required functional DAL81 and DAL82 gene products. Since the DAL81 product was not an allantoin pathway-specific regulatory factor, the DAL82 product was considered as the more likely candidate to be the DAL UIS binding protein. Using an E. coli expression system, we showed that DAL82 protein specifically bound to wild type but not mutant DAL UIS sequences. DNA fragments containing DAL UIS elements derived from various DAL gene promoters bound DAL82 protein with different affinities which correlate with the degree of inducer-responsiveness the genes displayed.

The broad-host-range plasmid pTF-FC2 requires a primase-like protein for autonomous replication in Escherichia coli.

A 3202-bp fragment of plasmid pTF-FC2, cloned into PUC19, had previously been identified as the minimum region required for replication in either Pseudomonas aeruginosa or Escherichia coli polA- mutants. During the course of experiments to construct broad-host-range cloning vectors based on the pTF-FC2 replicon, it was found that the 3202-bp fragment had an absolute requirement for some function of the pUC19 vector. This requirement was eliminated in the presence of co-resident pTF-FC2 derivatives. An additional 1239-bp fragment from pTF-FC2, immediately adjacent to the 3202-bp fragment, was identified which restored the ability of the pTF-FC2 replicon to replicate autonomously. Sequence analysis of the region revealed a single open reading frame encoding a 40-kDa polypeptide, which was synthesised in an in vitro transcription/translation system. A comparison of the amino acid sequence of this protein with sequence data banks revealed limited homology with the RepB' primase of the IncQ plasmid, RSF1010. An M13 delta lac 110 replication-deficient phage system was used to demonstrate that the 40-kDa protein did function as a primase with respect to replication at the origin of replication (vegetative) of pTF-FC2.

Characterization of the minimum replicon of the broad-host-range plasmid pTF-FC2 and similarity between pTF-FC2 and the IncQ plasmids.

The nucleotide sequence of a 3,202-base-pair fragment which contained the minimum region required for replication of the broad-host-range plasmid, pTF-FC2, has been determined. At least five open reading frames and a region that affected the host range were identified. Proteins corresponding in size and location to four of the five open reading frames were produced in an in vitro transcription-translation system. The predicted amino acid sequences of two of the proteins were aligned with those of the RepA and RepC proteins of the broad-host-range IncQ plasmid RSF1010 and found to be 43 and 60% homologous, respectively. Despite this similarity, neither the RepA nor the RepC protein of the IncQ plasmid was able to complement mutations in the pTF-FC2 repA and repC genes. Although there was a considerable amount of DNA homology between pTF-FC2 and RSF1010 in the oriV region and the region coding for the RepA and RepC proteins, no other homology between the two plasmids at either the DNA or protein level could be detected.

Identification and sequence of the basic replication region of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans.

The minimum region required for replication of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 in Escherichia coli was shown to be contained on a 2.05-kilobase fragment of DNA. A 184-base-pair fragment that was required in cis for plasmid replication was identified. This region was also involved in plasmid incompatibility. Nucleotide sequencing of this region revealed three perfectly conserved 22-base-pair tandemly repeated sequences. A comparison of this region with the equivalent region of the broad-host-range plasmid R1162 showed that the repeated sequences had 60% nucleotide homology. The 106-base-pair region immediately adjacent to the repeated sequences was 75% homologous. These plasmids were compatible.