Evolution of bacterial trp operons and their regulation

Survival and replication of most bacteria require the ability to synthesize the amino acid L-tryptophan whenever it is not available from the environment. In this article we describe the genes, operons, proteins, and reactions involved in tryptophan biosynthesis in bacteria, and the mechanisms they use in regulating tryptophan formation. We show that although the reactions of tryptophan biosynthesis are essentially identical, gene organization varies among species--from whole-pathway operons to completely dispersed genes. We also show that the regulatory mechanisms used for these genes vary greatly. We address the question--what are some potential advantages of the gene organization and regulation variation associated with this conserved, important pathway?     

Molecular characterization of propionyllysines in non-histone proteins

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones. The molecular components involved in the two protein modification pathways are unknown, hindering further functional studies. Here we report identification of the first three in vivo non-histone protein substrates of lysine propionylation in eukaryotic cells: p53, p300, and CREB-binding protein. We used mass spectrometry to map lysine propionylation sites within these three proteins. We also identified the first two in vivo eukaryotic lysine propionyltransferases, p300 and CREB-binding protein, and the first eukaryotic depropionylase, Sirt1. p300 was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation, like lysine acetylation, is a dynamic and regulatory post-translational modification. Based on these observations, it appears that some enzymes are common to the lysine propionylation and lysine acetylation regulatory pathways. Our studies therefore identified first several important players in lysine propionylation pathway.     

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.     

A novel acyl-CoA beta-transaminase characterized from a metagenome

Background

Bacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia.

Methodology/Principal Findings

We applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation.

Conclusions

We characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms.     

The repertoire of solute carriers of family 6: identification of new human and rodent genes

Tremendous amount of primary sequence information has been made available from the genome sequencing projects, although a complete annotation and identification of all genes is still far from being complete. Here, we present the identification of two new human genes from the pharmacologically important family of transporter proteins, solute carriers family 6 (SLC6). These were named SLC6A17 and SLC6A18 by HUGO. The human repertoire of SLC6 proteins now consists of 19 functional members and four pseudogenes. We also identified the corresponding orthologues and additional genes from mouse and rat genomes. Detailed phylogenetic analysis of the entire family of SLC6 proteins in mammals shows that this family can be divided into four subgroups. We used Hidden Markov Models for these subgroups and identified in total 430 unique SLC6 proteins from 10 animal, one plant, two fungi, and 196 bacterial genomes. It is evident that SLC6 proteins are present in both animals and bacteria, and that three of the four subfamilies of mammalian SLC6 proteins are present in Caenorhabditis elegans, showing that these subfamilies are evolutionary very ancient. Moreover, we performed tissue localization studies on the entire family of SLC6 proteins on a panel of 15 rat tissues and further, the expression of three of the new genes was studied using quantitative real-time PCR showing expression in multiple central and peripheral tissues. This paper presents an overall overview of the gene repertoire of the SLC6 gene family and its expression profile in rats.     

Evolution of metabolic pathways by chance assembly of enzyme proteins generated from sense and antisense strands of pre-existing genes.

In order to get an insight into the evolutionary aspect of metabolic pathways, especially of the ubiquitous glycolytic pathway, we have carried out an extensive search of sense-sense and sense-antisense similarities for enzyme proteins in the glycolytic pathway, the pentose phosphate cycle, alcohol and lactate fermentation pathways and the TCA cycle. This investigation of amino acid sequences reveals a curious pattern of similarity relations; no similarity can be found between the enzyme proteins in a section of the glycolytic pathway where the glyceraldehyde-3-phosphate or even glycerol-3-phosphate is converted into the pyruvate while many examples of sense-sense and sense-antisense similarities are found even between enzyme proteins in distant blocks, e.g. between the proteins in the TCA cycle and those in the pentose phosphate cycle, as well as between the functionally associated proteins in each of these blocks. Complementary to this characteristic pattern of amino acid sequence similarity, the search for similarities of nucleotide sequences also finds that the similarities of glycolytic enzyme genes, some sense-sense and others sense-antisense similarities, are concentrated on the nucleotide sequences of prokaryotic 16S or eukaryotic 18S ribosomal RNA gene with its flanks, although some of the copy sequences are also found in transfer RNA genes as well as in 23S or 26S ribosomal RNA gene. These results strongly suggest that the metabolic pathways have been developed by the chance assembly of enzyme proteins generated from the sense and antisense strands of pre-existing genes, e.g. the fermentation pathways and pentose phosphate cycle by the proteins from the genes of enzymes in the glycolytic pathway and the TCA cycle from all these successively increased genes, ascribing the origin of metabolic enzyme genes to the close relation between the glycolytic enzyme protein genes and the RNA gene cluster.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

CTnscr94, a conjugative transposon found in enterobacteria.

Conjugational transposons are important for horizontal gene transfer in gram-positive and gram-negative bacteria, but have not been reported yet for enteric bacteria. Salmonella senftenberg 5494-57 has previously been shown to transfer by conjugation genes for a sucrose fermentation pathway which were located on a DNA element called scr-94. We report here that the corresponding scr genes for a phosphoenolpyruvate-dependent sucrose:phosphotransferase system and a sucrose metabolic pathway are located on a large (ca. 100 kb) conjugative transposon renamed CTnscr94. The self-transmissible element integrates at two specific attachment sites in a RecA-independent way into the chromosome of Escherichia coli K-12 strains. One site was identified within pheV, the structural gene for a tRNA(Phe). Sequencing of both ends of CTnscr94 revealed the presence of the 3' part of pheV on one end such that after integration of the element, a complete pheV gene is retained. CTnscr94 represents, to our knowledge, the first conjugational transposon found in enteric bacteria.     

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.     

Distribution of genes for lysine biosynthesis through the aminoadipate pathway among prokaryotic genomes

Deinococcus radioduranshas homologous genes to the genes which from the Thermus: thermophilus gene cluster for lysine biosynthesis. Interestingly, those genes are clustered in Thermus, nevertheless they are scattered in Deinococcus. A similar gene cluster has only been found in Pyrococcus However, the phylogenetic analyses indicated that the deduced gene products from Deinococcus were the most closely related to the proteins encoded in the Thermus gene cluster for lysine biosynthesis. Therefore, those genes had not been transferred horizontally between Pyrococcus and Thermus. It is strongly suggested that a common ancestor of Deinococcus and Thermus possessed the genes for lysine biosynthesis through the aminoadipate pathway. These had been clustered through the evolution of Thermus or had been scattered from the gene cluster through the evolution of Deinococcus. In addition, I showed that LysW and its homologues were specialized proteins for the prokaryotic lysine biosynthesis through the aminoadipate pathway.     

1+1?=?3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.     

Functional analysis of immune response genes in Drosophila identifies JNK pathway as a regulator of antimicrobial peptide gene expression in S2 cells

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.     

Identification of a new expanding family of genes characterized by atypical LRR domains. Localization of a cluster preferentially expressed in oocyte

In the present work, we have used the in silico subtraction methodology to identify novel oocyte-specific genes in the mouse. By this way, we have identified in silico a new family of genes composed of more than 80 members. Sequence analysis showed that these genes belong to the superfamily of leucine-rich repeat (LRR) proteins. However, LRRs of this family display some variability in length and in amino acids composition within the beta-strands region, as more leucine residues are substituted by other hydrophobic amino acids as compared to canonical LRRs. Interestingly, for nine of these genes, the ESTs were represented almost exclusively in mouse egg libraries. Three of them were selected for experimental study. By RT-PCR and in situ hybridization, we confirmed their specific expression in the mouse oocyte from primary to preovulatory follicles. These three genes are localized in a cluster on mouse chromosome 4, in the vicinity of another recently discovered oocyte specific gene called oogenesin, that we also found to belong to the same family. We thus re-named this latter gene 'oogenesin-1', and the three genes identified here were named oogenesin-2, -3 and -4.