Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria.

Nucleotide metabolism and its control in lactic acid bacteria

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The virulence factors of this microorganism involved in colonization and the induction of lung lesions have been thoroughly studied and some have been well characterized. A. pleuropneumoniae binds preferentially to cells of the lower respiratory tract in a process involving different adhesins and probably biofilm formation. Apx toxins and lipopolysaccharides exert pathogenic effects on several host cells, resulting in typical lung lesions. Lysis of host cells is essential for the bacterium to obtain nutrients from the environment and A. pleuropneumoniae has developed several uptake mechanisms for these nutrients. In addition to persistence in lung lesions, colonization of the upper respiratory tract - and of the tonsils in particular - may also be important for long-term persistent asymptomatic infection. Information on virulence factors involved in tonsillar and nasal cavity colonization and persistence is scarce, but it can be speculated that similar features as demonstrated for the lung may play a role. Actinobacillus pleuropneumoniae / virulence factor / pig / respiratory disease

https://biblio.ugent.be/publication/1182005/file/1182037.pdf

Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host.

ClpP proteins constitute a family of homologous proteins found in both prokaryotic and eukaryotic organisms. In Escherichia coli, ClpP is the proteolytic component of a large complex also containing either the ClpA or the ClpX ATPases. We show here that the clpP gene from the Gram-positive bacterium Lactococcus lactis encodes a 22-kDa protein that is induced by low pH and by the t-RNA analogue puromycin, which interferes with translation, resulting in the production of misfolded puromycyl-containing peptides. Northern blot and primer extension analysis showed that clpP expression is also induced by heat shock and that stress induction occurs at the transcriptional level independent of the CIRCE regulatory element often implicated in stress regulation in Gram-positive bacteria. When we disrupted the L. lactis clpP gene by insertional inactivation, the resulting mutant was more sensitive to both heat and puromycin than wild-type cells. Furthermore, cells lacking ClpP had a reduced ability to degrade puromycyl-containing peptides, and they synthesized heat shock proteins constitutively in the absence of stress. Thus, our data suggest that ClpP plays a major role in the degradation of misfolded proteins.

ClpP participates in the degradation of misfolded protein in Lactococcus lactis

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood.

Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

Serotype-specific DNA regions involved in the biosynthesis of capsular polysaccharides (cps region) were used to develop a multiplex PCR test for the simultaneous species identification and serotyping of Actinobacillus pleuropneumoniae serotypes 2, 5, and 6. Primers specific for serotypes 2, 5, and 6 were combined with the already existing species-specific primers used in a PCR test based on the omlA gene. The PCR test was evaluated with serotype reference strains of A. pleuropneumoniae as well as 182 Danish field isolates previously serotyped by latex agglutination or immunodiffusion. For all serologically typeable strains, a complete correspondence was found between the results obtained by the multiplex PCR test and the results obtained by the traditional serotyping methods. Six of eight serologically nontypeable strains could be allocated to a serotype on the basis of the multiplex PCR results. The species specificity of the assay was evaluated with a collection of 93 strains representing 29 different species within the family Pasteurellaceae, as well as species normally found in the respiratory tracts of swine. All of these strains were negative by the multiplex PCR test, including 50 field isolates of the phylogenetically closely related species Actinobacillus lignieresii. When the multiplex PCR test was used to test Danish field strains, it was able to identify the serotypes of approximately 94% of all strains isolated from swine with clinical disease. More than 90% of the isolates that cross-reacted by the latex agglutination test were of serotype 2, 5, or 6. Determination of the serotype by PCR represents a convenient and specific method for the serotyping of A. pleuropneumoniae in diagnostic laboratories.

Evaluation of a Multiplex PCR Test for Simultaneous Identification and Serotyping of Actinobacillus pleuropneumoniae Serotypes 2, 5, and 6

Multiplex PCR assays were developed to identify Actinobacillus pleuropneumoniae serotypes 1, 2, and 8. Primers designed for the conserved capsular polysaccharide (CP) export region amplified a 489-bp DNA fragment from all serotypes. Primers specific to the CP biosynthesis regions of serotypes 1, 2, and 8 amplified fragments of 1.6 kb, 1.7 kb, and 970 bp from only their respective serotypes.

Detection and identification of Actinobacillus pleuropneumoniae serotypes 1, 2, and 8 by multiplex PCR.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the purC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea that purC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

Activation Control of pur Gene Expression in Lactococcus lactis: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of purC and purD Promoter Regions

Stine Graakjær Jessing

Papers

Evaluation of a Multiplex PCR Test for Simultaneous Identification and Serotyping of Actinobacillus pleuropneumoniae Serotypes 2, 5, and 6

Detection and identification of Actinobacillus pleuropneumoniae serotypes 1, 2, and 8 by multiplex PCR.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

Activation Control of pur Gene Expression in Lactococcus lactis: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of purC and purD Promoter Regions

The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12.