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PubMed2006

Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates.

BACKGROUND: Evolution of the deuterostome lineage was accompanied by an increase in systematic complexity especially with regard to highly specialized tissues and organs. Based on the observation of an increased number of paralogous genes in vertebrates compared with invertebrates, two entire genome duplications (2R) were proposed during the early evolution of vertebrates. Most glycolytic enzymes occur as several copies in vertebrate genomes, which are specifically expressed in certain tissues. Therefore, the glycolytic pathway is particularly suitable for testing theories of the involvement of gene/genome duplications in enzyme evolution. RESULTS: We assembled datasets from genomic databases of at least nine vertebrate species and at least three outgroups (one deuterostome and two protostomes), and used maximum likelihood and Bayesian methods to construct phylogenies of the 10 enzymes of the glycolytic pathway. Through this approach, we intended to gain insights into the vertebrate specific evolution of enzymes of the glycolytic pathway. Many of the obtained gene trees generally reflect the history of two rounds of duplication during vertebrate evolution, and were in agreement with the hypothesis of an additional duplication event within the lineage of teleost fish. The retention of paralogs differed greatly between genes, and no direct link to the multimeric structure of the active enzyme was found. CONCLUSION: The glycolytic pathway has subsequently evolved by gene duplication and divergence of each constituent enzyme with taxon-specific individual gene losses or lineage-specific duplications. The tissue-specific expression might have led to an increased retention for some genes since paralogs can subdivide the ancestral expression domain or find new functions, which are not necessarily related to the original function.

Animals↗
PubMed2002

Selection in the evolution of gene duplications.

BACKGROUND: Gene duplications have a major role in the evolution of new biological functions. Theoretical studies often assume that a duplication per se is selectively neutral and that, following a duplication, one of the gene copies is freed from purifying (stabilizing) selection, which creates the potential for evolution of a new function. RESULTS: In search of systematic evidence of accelerated evolution after duplication, we used data from 26 bacterial, six archaeal, and seven eukaryotic genomes to compare the mode and strength of selection acting on recently duplicated genes (paralogs) and on similarly diverged, unduplicated orthologous genes in different species. We find that the ratio of nonsynonymous to synonymous substitutions (Kn/Ks) in most paralogous pairs is <<1 and that paralogs typically evolve at similar rates, without significant asymmetry, indicating that both paralogs produced by a duplication are subject to purifying selection. This selection is, however, substantially weaker than the purifying selection affecting unduplicated orthologs that have diverged to the same extent as the analyzed paralogs. Most of the recently duplicated genes appear to be involved in various forms of environmental response; in particular, many of them encode membrane and secreted proteins. CONCLUSIONS: The results of this analysis indicate that recently duplicated paralogs evolve faster than orthologs with the same level of divergence and similar functions, but apparently do not experience a phase of neutral evolution. We hypothesize that gene duplications that persist in an evolving lineage are beneficial from the time of their origin, due primarily to a protein dosage effect in response to variable environmental conditions; duplications are likely to give rise to new functions at a later phase of their evolution once a higher level of divergence is reached.

Animals↗
PubMed2005

Localization, structure and polymorphism of two paralogous Xenopus laevis mitochondrial malate dehydrogenase genes.

Two paralogous mitochondrial malate dehydrogenase 2 (Mdh2) genes of Xenopus laevis have been cloned and sequenced, revealing 95% identity. Fluorescence in-situ hybridization (FISH) combined with tyramide amplification discriminates both genes; Mdh2a was localized into chromosome q3 and Mdh2b into chromosome q8. One kb cDNA probes detect both genes with 85% accuracy. The remaining signals were on the paralogous counterpart. Introns interrupt coding sequences at the same nucleotide as defined for mouse. Restriction polymorphism has been detected in the first intron of Mdh2a, while the individual variability in intron 6 of Mdh2b gene is represented by an insertion of incomplete retrotransposon L1Xl. Rates of nucleotide substitutions indicate that both genes are under similar evolutionary constraints. X. laevis Mdh2 genes can be used as markers for physical mapping and linkage analysis.

Amino Acid Sequence↗
PubMed2003

MHC class I-like genes in cattle, MHCLA, with similarity to genes encoding NK cell stimulatory ligands.

A comparative genomics approach for mining databases of expressed sequence tags (ESTs) was used to identify two members of a novel MHC class I gene family in cattle. These paralogous genes, named MHC class I-like gene family A1 ( MHCLA1) and MHCLA2, were shown by phylogenetic analysis to be related to human and mouse genes encoding NK cell stimulatory ligands, ULBP, RAET, H60 and Raet-1. Radiation hybrid mapping placed cattle MHCLA1 on BTA9, which, on the basis of existing comparative mapping data, identified the ULBP, RAET1, H60 and Raet1 genes as homologues of the cattle MHCLA genes. However, the human and mouse orthologues of MHCLA1 and MHCLA2 could not be defined due to extensive sequence divergence from all known members of the ULBP1/ RAET1/H60/Raet1 gene family. The cattle MHCLA1 molecule is predicted to be missing an alpha(3) domain, similar to the human and mouse homologues. Like the human ULBP genes, MHCLA1 was found to be transcribed constitutively in a variety of fetal and adult tissues by RT-PCR. The patterns of hybridization obtained by Southern blotting using MHCLA1 as a probe and DNA from 14 species representing five mammalian orders suggests that the MHCLA genes evolved rapidly in the Cetartiodactyla. Previous findings demonstrating that ULBPs serve as ligands for the NK cell NKG2D stimulatory receptor, and that this interaction can be blocked by a human cytomegalovirus glycoprotein that binds to ULBPs, suggests that the extensive divergence found among the cattle, human and mouse MHCLA homologues is due to selection exerted by viral pathogens.

Amino Acid Sequence↗
PubMed2004

Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.

Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions.

Amino Acid Sequence↗
PubMed2006

Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair.

BACKGROUND: The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability. RESULTS: Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains. CONCLUSION: This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.

Evolution, Molecular↗
PubMed1994

Axial homeosis and appendicular skeleton defects in mice with a targeted disruption of hoxd-11.

Using gene targeting, we have created mice with a disruption in the homeobox-containing gene hoxd-11. Homozygous mutants are viable and the only outwardly apparent abnormality is male infertility. Skeletons of mutant mice show a homeotic transformation that repatterns the sacrum such that each vertebra adopts the structure of the next most anterior vertebra. Defects are also seen in the bones of the limb, including regional malformations at the distal end of the forelimb affecting the length and structure of phalanges and metacarpals, inappropriate fusions between wrist bones, and defects at the most distal end in the long bones of the radius and ulna. The phenotypes show both incomplete penetrance and variable expressivity. In contrast to the defects observed in the vertebral column, the phenotypes in the appendicular skeleton do not resemble homeotic transformations, but rather regional malformations in the shapes, length and segmentation of bones. Our results are discussed in the context of two other recent gene targeting studies involving the paralogous gene hoxa-11 and another member of the Hox D locus, hoxd-13. The position of these limb deformities reflects the temporal and structural colinearity of the Hox genes, such that inactivation of 3' genes has a more proximal phenotypic boundary (affecting both the zeugopod and autopod of the limb) than that of the more 5' genes (affecting only the autopod). Taken together, these observations suggest an important role for Hox genes in controlling localized growth of those cells that contribute to forming the appendicular skeleton.

Animals↗
PubMed2004

Recolonization and radiation in Larix (Pinaceae): evidence from nuclear ribosomal DNA paralogues.

Gene paralogy frequently causes the conflict between gene tree and species tree, but sometimes the coexistence of a few paralogous copies could provide more markers for tracing the phylogeographical process of some organisms. In the present study, nrDNA ITS paralogues were cloned from all but one species of Larix, an Eocene genus having two sections, Larix and Multiserialis, with a huge circumboreal distribution and an Eastern Asia-Western North America disjunction, respectively. A total of 96 distinct clones, excluding five putative pseudogenes or recombinants, were obtained and used in the gene genealogy analysis. The clones from all Eurasian species of section Larix are mixed together, suggesting that recolonization and recent morphological differentiation could have played important roles in the evolution of this section. In contrast, the species diversification of the Eurasian section Multiserialis may result from radiation in the east Himalayas and its vicinity, considering extensive nrDNA founder effects in this group. Our study also suggests that the distribution pattern analysis of members of multiple gene family would be very useful in tracking the evolutionary history of some taxa with recent origin or rapid radiation that cannot be resolved by other molecular markers.

Base Sequence↗
PubMed2001

The one-to-four rule and paralogues of sex-determining genes.

Because of two successive rounds of tetraploidization at their inception, the vertebrates contain four times more protein-coding genes in their genome than the invertebrates: 60,000 vs. 15,000. Consequently, each invertebrate gene has been amplified to the maximum of four paralogous genes in vertebrates: the one-to-four rule. When this rule is applied to genes pertinent to gonadal development and differentiation, the following emerged: (i) Two closely related zinc-finger transcription factor genes in invertebrates have been amplified to two paralogous groups in vertebrates. One consisted of EGR1, EGR2, EGR3 and EGR4, whereas the only known paralogue of the other is WT1, which controls the developmental fate of the entire nephric system, and therefore of gonads. Interestingly, EGR1 and WT1 act as antagonists of each other in nephroblastic cells. (ii) SF-1, which controls the fate of two steroid hormone-producing organs, adrenals and gonads, is descended from the invertebrate Ftz-F1 gene, and its only known paralogue is GCNF-1. (iii) The Y-linked SRY, the mammalian testis-determining gene, is a paralogue neither of SOX3 (SRX) nor of SOX9. Its ancient origin suggests that SRY once became extinct in earlier vertebrates, only to revive itself in the mammalian ancestor. (iv) Inasmuch as four paralogues of one invertebrate nuclear receptor gene have differentiated to receptors of androgen, mineralocorticoid, glucocorticoid and progesterone, there should at most be four paralogous estrogen-receptor genes in the vertebrate genome. It is likely that one of them plays a pivotal role in the estrogen-dependent sex-determining mechanism so commonly found among reptiles, amphibians and fish.

Animals↗
PubMed1999

The one-to-four rule and paralogues of sex-determining genes.

Because of two successive rounds of tetraploidization at their inception, the vertebrates contain four times more protein-coding genes in their genome than the invertebrates: 60,000 versus 15,000. Consequently, each invertebrate gene has been amplified to the maximum of four paralogous genes in vertebrates: the one-to-four rule. When this rule is applied to genes pertinent to gonadal development and differentiation, the following emerged: (i) Two closely related zinc-finger transcription factor genes in invertebrates have been amplified to two paralogous groups in vertebrates. One consisted of EGR1, EGR2, EGR3 and EGR4, whereas the only known paralogue of the other is WT1, which controls the developmental fate of the entire nephric system, and therefore of gonads. Interestingly, EGR1 and WT1 act as antagonists of each other in nephroblastic cells. (ii) SF-1, which controls the fate of two steroid hormone-producing organs, adrenals and gonads, is descended from the invertebrate Ftz-F1 gene, and its only known paralogue is GCNF-1. (iii) The Y-linked SRY, the mammalian testis-determining gene, is a paralogue neither of SOX3 (SRX) nor of SOX9. Its ancient origin suggests that SRY once became extinct in earlier vertebrates, only to revive itself in the mammalian ancestor. (iv) Inasmuch as four paralogues of one invertebrate nuclear receptor gene have differentiated to receptors of androgen, mineralocoticoid, glucocorticoid and progesterone, there should at most be four paralogous estrogen-receptor genes in the vertebrate genome. It is likely that one of them plays a pivotal role in the estrogen-dependent sex-determining mechanism so commonly found among reptiles, amphibians and fish.

Adrenal Cortex↗
PubMed2006

Evolutionary fate of duplicate genes encoding aspartic proteinases. Nothepsin case study.

Gene duplication is considered an important evolutionary mechanism leading to new gene functions. According to the classical model, one gene copy arising from gene duplication retains the ancestral function, whilst the other becomes subject to directional selection for some novel functions. Hence, according to this model, long-term persistence of two paralogous genes is possible only with the acquisition of functional innovation. In the absence of neofunctionalization, one of the duplicate genes may be lost following accumulation of deleterious mutations, ultimately leading to the loss of function. Recently, new mechanisms have been proposed according to which both paralogs are maintained without apparent neofunctionalization. In this paper we describe the molecular evolution of the aspartic proteinase gene family, with particular regard for the nothepsin gene, a sex- and tissue-specific form of aspartic proteinase active in fish. The finding of nothepsin in a reptile is indicative of the presence of this gene in organisms other than fish. However, the failure to find any nothepsin-like gene in avian, murine and human genome suggests that the gene has been lost in certain lineages during evolution. At variance with piscine nothepsin expressed exclusively in female liver under the estrogens action, the reptilian counterpart lacks both tissue and sex specificity, as it is constitutively expressed in different tissues of male and female specimens. The expression of the nothepsin gene in fish and lizard is accompanied by the expression of a paralogous gene encoding for cathepsin D. Functional divergence analysis indicates that cathepsin D accumulated amino acid substitutions, whereas nothepsin retained most of the ancestral functions. Phylogenetic analysis shows a preponderance of replacement substitutions compared to silent substitutions in the branch leading to the cathepsin D clade, whilst nothepsin evolves under negative selection. To explain the loss of the nothepsin gene in certain lineages, we propose a model that takes into account the complementary degenerative mutations occurring in regulatory elements of the promoter regions of the two genes. According to this model, gene loss occurs whenever the two genes acquire the same expression pattern. The coexistence of cathepsin D and nothepsin is explained in terms of metabolic cooperation of the two enzymes.

Amino Acid Sequence↗
PubMed1999

Recurrent paralogy in the evolution of archaeal chaperonins.

Chaperonins are multisubunit double-ring complexes that mediate the folding of nascent proteins [1] [2]. In bacteria, chaperonins are homo-oligomeric and are composed of seven-membered rings. Eukaryotic and most archaeal chaperonin rings are eight-membered and exhibit varying degrees of hetero-oligomerism [3] [4]. We have cloned and sequenced seven new genes encoding chaperonin subunits from the crenarchaeotes Sulfolobus solfataricus, S. acidocaldarius, S. shibatae and Desulfurococcus mobilis. Although some archaeal genomes possess a single chaperonin gene, most have two. We describe a third chaperonin-encoding gene (TF55-gamma) from two Sulfolobus species; phylogenetic analyses indicate that the gene duplication producing TF55-gamma occurred within crenarchaeal evolution. The presence of TF55-gamma in Sulfolobus correlates with their unique nine-membered chaperonin rings. Duplicate genes (paralogs) for chaperonins within archaeal genomes very often resemble each other more than they resemble chaperonin genes from other archaea. Our phylogenetic analyses suggest multiple independent gene duplications - at least seven among the archaea examined. The persistence of paralogous genes for chaperonin subunits in multiple archaeal lineages may involve a process of co-evolution, where chaperonin subunit heterogeneity changes independently of selection on function.

Archaea↗
PubMed1999

Identification of three novel Ca(2+) channel gamma subunit genes reveals molecular diversification by tandem and chromosome duplication.

Gene duplication is believed to be an important evolutionary mechanism for generating functional diversity within genomes. The accumulated products of ancient duplication events can be readily observed among the genes encoding voltage-dependent Ca(2+) ion channels. Ten paralogous genes have been identified that encode isoforms of the alpha(1) subunit, four that encode beta subunits, and three that encode alpha(2)delta subunits. Until recently, only a single gene encoding a muscle-specific isoform of the Ca(2+) channel gamma subunit (CACNG1) was known. Expression of a distantly related gene in the brain was subsequently demonstrated upon isolation of the Cacng2 gene, which is mutated in the mouse neurological mutant stargazer (stg). In this study, we sought to identify additional genes that encoded gamma subunits. Because gene duplication often generates paralogs that remain in close syntenic proximity (tandem duplication) or are copied onto related daughter chromosomes (chromosome or whole-genome duplication), we hypothesized that the known positions of CACNG1 and CACNG2 could be used to predict the likely locations of additional gamma subunit genes. Low-stringency genomic sequence analysis of targeted regions led to the identification of three novel Ca(2+) channel gamma subunit genes, CACNG3, CACNG4, and CACNG5, on chromosomes 16 and 17. These results demonstrate the value of genome evolution models for the identification of distantly related members of gene families.

Amino Acid Sequence↗
PubMed1998

A third human CBL gene is on chromosome 19.

CBL genes encode cytoplasmic proteins involved in signal transduction downstream of a number of receptors including tyrosine kinases, cytokine receptors, and T-cell or B-cell receptors. They seem to be transducers associated with negative regulation of signals, and, as such, may be potential tumor suppressors. Using a probe derived from an expressed sequence tag, we isolated a cosmid containing part of a new CBL gene, CBLc, related to the two characterized paralogous genes CBLa and CBLb. Using the cosmid in fluorescence in situ hybridization of human metaphase chromosomes, we localized the CBLc gene to band 13.2 of chromosome 19. We show that the 19q12.2-13.3 region where CBLc is located shows paralogy with two other regions of the human genome, 3q22-q27 and 11q22-q24 where CBLb and CBLa are located, respectively. Genes from several other families are located in these regions.

Chromosome Mapping↗
PubMed2005

Functional genomics of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1.

Nitrate-reducing bacteria of the recently recognized Azoarcus/Thauera group within the Betaproteobacteria contribute significantly to the biodegradation of aromatic and other refractory compounds in anoxic waters and soils. Strain EbN1 belongs to a distinct cluster (new genus) and is the first member of this phylogenetic group, the genome of which has been determined (4.7 Mb; one chromosome, two plasmids) by [Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27-36]. Ten anaerobic and four aerobic aromatic-degradation pathways were recognized on the chromosome, with the coding genes mostly forming clusters. Presence of paralogous gene clusters (e.g. for anaerobic ethylbenzene degradation) suggests an even broader degradation spectrum than previously known. Metabolic versatility is also reflected by the presence of multiple respiratory complexes and is apparently controlled by an extensive regulatory network. Strain EbN1 is unique for its capacity to degrade toluene and ethylbenzene anaerobically via completely different pathways. Bioinformatical analysis of their genetic blueprints and global expression analysis (DNA-microarray and proteomics) of substrate-adapted cells [Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus R (2005) Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187:1493-1503] indicated coordinated vs sequential modes of regulation for the toluene and ethylbenzene pathways, respectively.

Anaerobiosis↗
PubMed1994

Targeted disruptions of the murine Hoxa-4 and Hoxa-6 genes result in homeotic transformations of components of the vertebral column.

It is becoming clear that Hox genes, which encode transcription factors of the Antennapedia homeodomain family, are key players in establishing the body plan of mammalian embryos. They have already been implicated in the formation of the central nervous system, tissues derived from neural crest, the vertebral column and the limbs. In order to examine the roles of hoxa-4 and hoxa-6 during development, mice with targeted disruptions in these genes were generated. Each shows homeotic transformation of cervical vertebrae, at positions that approximate the anterior borders of expression of these genes in the prevertebrae. Defects were not observed in other tissues that normally express these genes. Hoxa-4-/hoxa-4- mice show, with 100% penetrance, anterior transformations of the dorsal aspects of the third cervical vertebra by acquiring features normally associated with the second cervical vertebra. Mice homozygous for the hoxa-6 mutation show, with incomplete penetrance, even on opposite sides of the same animal, posterior transformations of the seventh cervical vertebra to the first thoracic vertebra. In addition, both hoxa-4-/hoxa-4- and hoxa-6-/hoxa-6- mice show variability in expressivity. These data indicate that alternative genetic pathways can partially, and at times completely, substitute for the function of these two genes. Other members of these two paralogous Hox families are good candidates for providing the substitutions. As paralogous genes lie on different chromosomes, it is possible to examine the degree of redundancy among these genes by intercrossing mice with the appropriate individual disruptions. The analysis of double, triple and even quadruple mutants should determine the ways in which these Hox genes interact in order to specify the multitude of tissues in a restricted region of the developing mouse embryo.

Alleles↗
PubMed1998

Homology-dependent gene silencing in Paramecium.

Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant.

Animals↗
PubMed2003

Genome engineering reveals large dispensable regions in Bacillus subtilis.

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.

Bacillus Phages↗