Amino Acid metabolism conflicts with protein diversity.

The 20 protein-coding amino acids are found in proteomes with different relative abundances. The most abundant amino acid, leucine, is nearly an order of magnitude more prevalent than the least abundant amino acid, cysteine. Amino acid metabolic costs differ similarly, constraining their incorporation into proteins. On the other hand, a diverse set of protein sequences is necessary to build functional proteomes. Here, we present a simple model for a cost-diversity trade-off postulating that natural proteomes minimize amino acid metabolic flux while maximizing sequence entropy. The model explains the relative abundances of amino acids across a diverse set of proteomes. We found that the data are remarkably well explained when the cost function accounts for amino acid chemical decay. More than 100 organisms reach comparable solutions to the trade-off by different combinations of proteome cost and sequence diversity. Quantifying the interplay between proteome size and entropy shows that proteomes can get optimally large and diverse.

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria.

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition-fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes.

A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette.

Since its initial description more than two decades ago, the ribosome bypass (or "hop") sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an in-phase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron.

A likely pathway for formation of mobile group I introns.

Mobile group I introns are RNA splicing elements that have been invaded by endonuclease genes. These endonucleases facilitate intron mobility by a unidirectional, duplicative gene-conversion process known as homing [1]. Survival of the invading endonuclease depends upon its ability to promote intron mobility. Therefore, the endonuclease must either quickly change its cleavage specificity to match the site of intron insertion, or it must already be preadapted to cleave this sequence. Here we show that the group I intron in the DNA polymerase gene of T7-like bacteriophage PhiI is mobile, dependent upon its intronic HNH homing endonuclease gene, I-TslI. We also show that gene 5.3 of phage T3, located adjacent to its intronless DNA polymerase gene, is a homologous homing endonuclease gene whose protein product initiates efficient spread of gene 5.3 into empty sites in related phages. Both of these endonucleases cleave intronless DNA polymerase genes at identical positions. This shared feature between an intronic and free-standing endonuclease is unprecedented. Based on this evidence, we propose that introns and their homing endonucleases evolve separately to target the same highly conserved sequences, uniting afterwards to create a composite mobile element.

A free-standing homing endonuclease targets an intron insertion site in the psbA gene of cyanophages.

Homing endonuclease genes are mobile elements that promote their duplication into cognate sites that lack the endonuclease gene [1, 2]. The homing endonuclease initiates this event through site-specific DNA cleavage. Copying of the endonuclease gene follows as a consequence of DNA repair. A genome containing a homing endonuclease gene is subject to self-cleavage. Protection is accomplished through DNA sequence polymorphisms, as is the case in intronless homing of free-standing endonuclease genes [3, 4], or by disruption of the recognition site by a group I intron (or intein) into which the endonuclease ORF is embedded. We describe here a novel free-standing homing endonuclease from cyanobacteriophage S-PM2, which is similar to the DNA resolvase of bacteriophage T4 and is encoded adjacent to an intron-containing psbA gene [5, 6]. The endonuclease makes a specific double-strand cut near the intron insertion site (IIS), its DNA recognition site spans the IIS, and it is unable to cleave intron-containing psbA genes. This interdependence of a free-standing endonuclease gene and a group I intron, which we denote "collaborative homing," has not been reported previously and gives support to a hypothesis of formation of composite mobile introns by independent convergence of an intron and an endonuclease gene on the same target sequence.

The restriction fold turns to the dark side: a bacterial homing endonuclease with a PD-(D/E)-XK motif.

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene-the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease - a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases - which in turn, are rarely incorporated into restriction/modification systems.

I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities.

I-HmuI and I-BasI are two highly similar nicking DNA endonucleases, which are each encoded by a group I intron inserted into homologous sites within the DNA polymerase genes of Bacillus phages SPO1 and Bastille, respectively. Here, we present a comparison of the DNA specificities and cleavage activities of these enconucleases with homologous target sites. I-BasI has properties that are typical of homing endonucleases, nicking the intron-minus polymerase genes in either host genome, three nucleotides downstream of the intron insertion site. In contrast, I-HmuI nicks both the intron-plus and intron-minus site in its own host genome, but does not act on the target from Bastille phage. Although the enzymes have distinct DNA substrate specificities, both bind to an identical 25bp region of their respective intron-minus DNA polymerase genes surrounding the intron insertion site. The endonucleases appear to interact with the DNA substrates in the downstream exon 2 in a similar manner. However, whereas I-HmuI is known to make its only base-specific contacts within this exon region, structural modeling analyses predict that I-BasI might make specific base contacts both upstream and downstream of the site of intron insertion. The predicted requirement for base-specific contacts in exon 1 for cleavage by I-BasI was confirmed experimentally. This explains the difference in substrate specificities between the two enzymes, including the observation that the former enzyme is relatively insensitive to the presence of an intron upstream of exon 2. These differences are likely a consequence of divergent evolutionary constraints.

A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages.

Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of PhiI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.

Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor.

Customary binding sites of intron-encoded homing endonucleases lie within cognate intronless alleles, at the so-called homing sites. Here, we describe a novel, high-affinity binding site for I-TevI endonuclease, encoded within the group I td intron of phage T4. This site is an operator that overlaps the T4 late promoter, which drives I-TevI expression from within the td intron. I-TevI binds the operator and homing sites with equal affinity, and functions as a transcriptional autorepressor. Distinct sequence and spacing requirements of the catalytic domain result in reduced cleavage activity on operator DNA. Crystallographic studies showed that the overall interactions of the DNA-binding domain with the operator and homing sites are similar, but have some different hydrogen-bonding contacts. We present a model in which the flexibility in protein-DNA interactions allows I-TevI to bind variant intronless alleles to promote intron mobility while facilitating its function in autorepression, and thereby persistence in its host.

DNA binding and cleavage by the HNH homing endonuclease I-HmuI.

The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.

Genetic organization of the psbAD region in phages infecting marine Synechococcus strains.

The discovery of the genes psbA and psbD, encoding the D1 and D2 core components of the photosynthetic reaction center PSII (photosystem II), in the genome of the bacteriophage S-PM2 (a cyanomyovirus) that infects marine cyanobacteria begs the question as to how these genes were acquired. In an attempt to answer this question, it was established that the occurrence of the genes is widespread among marine cyanomyovirus isolates and may even extend to podoviruses. The phage psbA genes fall into a clade that includes the psbA genes from their potential Synechococcus and Prochlorococcus hosts, and thus, this phylogenetic analysis provides evidence to support the idea of the acquisition of these genes by horizontal gene transfer from their cyanobacterial hosts. However, the phage psbA genes form distinct subclades within this lineage, which suggests that their acquisition was not very recent. The psbA genes of two phages contain identical 212-bp insertions that exhibit all of the canonical structural features of a group I self-splicing intron. The different patterns of genetic organization of the psbAD region are consistent with the idea that the psbA and psbD genes were acquired more than once by cyanomyoviruses and that their horizontal transfer between phages via a common phage gene pool, as part of mobile genetic modules, may be a continuing process. In addition, genes were discovered encoding a high-light inducible protein and a putative key enzyme of dark metabolism, transaldolase, extending the areas of host-cell metabolism that may be affected by phage infection.

Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease.

Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.

SegG endonuclease promotes marker exclusion and mediates co-conversion from a distant cleavage site.

Bacteriophages T2 and T4 are closely related T-even phages. However, T4 genetic markers predominate in the progeny of mixed infections, a phenomenon termed marker exclusion. One region previously mapped where the frequency of T2 markers in the progeny is extremely low is located around gene 32. Here, we describe SegG, a GIY-YIG family endonuclease adjacent to gene 32 of phage T4 that is absent from phage T2. In co-infections with T2 and T4, cleavage in T2 gene 32 by T4-encoded SegG initiates a gene conversion event that results in replacement of T2 gene 32 markers with the corresponding T4 sequence. Interestingly, segG inheritance is limited, apparently because of the physical separation of its cleavage and insertion sites, which are 332 base-pairs apart. This contrasts with efficient inheritance of the phage T4 td group I intron and its endonuclease, I-TevI, for which the distance separating the I-TevI cleavage site and td insertion site is 23 base-pairs. Furthermore, we show that co-conversion tracts generated by repair of SegG and I-TevI double-strand breaks contribute to the localized exclusion of T2 markers. Our results demonstrate that the endonuclease activities of SegG and I-TevI promote the spread of these two endonucleases to progeny phage, consistent with their role as selfish genetic elements, and also provide a mechanism by which the genetic contribution of T2 markers to progeny phage is reduced.

The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille.

Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille. Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure. Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI. Unlike I-HmuI, which nicks both intron-minus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing endonucleases. Interestingly, the C-terminal portions of these H-N-H phage endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular betabetaalphaalphabeta fold, suggestive of module shuffling between different homing endonuclease families.

Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort.

We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome. Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE). Reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from Staphylococcus aureus after phage infection indicates that the introns are removed from the primary transcript in vivo. Both nrdE introns show sequence similarity to the Twort orf142 introns I2 and I3, suggesting either a common origin of these introns or shuffling of intron structural elements. Intron 2 encodes a DNA endonuclease, I-TwoI, with similarity to homing endonucleases of the HNH family. Like I-HmuI and I-HmuII, intron-encoded HNH endonucleases in Bacillus subtilis phages SPO1 and SP82, I-TwoI nicks only one strand of its DNA recognition sequence. However, whereas I-HmuI and I-HmuII cleave the template strand in exon 2, I-TwoI cleaves the coding strand in exon 1. In each case, the 3' OH created on the cut strand is positioned to prime DNA synthesis towards the intron, suggesting that this reaction contributes to the mechanism of intron homing. Both nrdE introns are inserted in highly conserved regions of the ribonucleotide reductase gene, next to codons for functionally important residues.

Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only approximately 1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events.