A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications.     

Optimal gene partition into operons correlates with gene functional order

Gene arrangement into operons varies between bacterial species. Genes in a given system can be on one operon in some organisms and on several operons in other organisms. Existing theories explain why genes that work together should be on the same operon, since this allows for advantageous lateral gene transfer and accurate stoichiometry. But what causes the frequent separation into multiple operons of co-regulated genes that act together in a pathway? Here we suggest that separation is due to benefits made possible by differential regulation of each operon. We present a simple mathematical model for the optimal distribution of genes into operons based on a balance of the cost of operons and the benefit of regulation that provides 'just-when-needed' temporal order. The analysis predicts that genes are arranged such that genes on the same operon do not skip functional steps in the pathway. This prediction is supported by genomic data from 137 bacterial genomes. Our work suggests that gene arrangement is not only the result of random historical drift, genome re-arrangement and gene transfer, but has elements that are solutions of an evolutionary optimization problem. Thus gene functional order may be inferred by analyzing the operon structure across different genomes.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes

The Selfish Operon Model postulates that the organization of bacterial genes into operons is beneficial to the constituent genes in that proximity allows horizontal cotransfer of all genes required for a selectable phenotype; eukaryotic operons formed for very different reasons. Horizontal transfer of selfish operons most probably promotes bacterial diversification.     

Experimental demonstration of operon formation catalyzed by insertion sequence

Operons are a hallmark of the genomic and regulatory architecture of prokaryotes. However, the mechanism by which two genes placed far apart gradually come close and form operons remains to be elucidated. Here, we propose a new model of the origin of operons: Mobile genetic elements called insertion sequences can facilitate the formation of operons by consecutive insertion–deletion–excision reactions. This mechanism barely leaves traces of insertion sequences and thus difficult to detect in nature. In this study, as a proof-of-concept, we reproducibly demonstrated operon formation in the laboratory. The insertion sequence IS3 and the insertion sequence excision enhancer are genes found in a broad range of bacterial species. We introduced these genes into insertion sequence-less Escherichia coli and found that, supporting our hypothesis, the activity of the two genes altered the expression of genes surrounding IS3, closed a 2.7 kb gap between a pair of genes, and formed new operons. This study shows how insertion sequences can facilitate the rapid formation of operons through locally increasing the structural mutation rates and highlights how coevolution with mobile elements may shape the organization of prokaryotic genomes and gene regulation.     

Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa

Bacteriophage B3 is a transposable phage of Pseudomonas aeruginosa. In this report, we present the complete DNA sequence and annotation of the B3 genome. DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3%. The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons. Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins. Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes. Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions. Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster. These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.     

Genome holography: deciphering function-form motifs from gene expression data

Background

DNA chips allow simultaneous measurements of genome-wide response of thousands of genes, i.e. system level monitoring of the gene-network activity. Advanced analysis methods have been developed to extract meaningful information from the vast amount of raw gene-expression data obtained from the microarray measurements. These methods usually aimed to distinguish between groups of subjects (e.g., cancer patients vs. healthy subjects) or identifying marker genes that help to distinguish between those groups. We assumed that motifs related to the internal structure of operons and gene-networks regulation are also embedded in microarray and can be deciphered by using proper analysis.

Methodology/Principal Findings

The analysis presented here is based on investigating the gene-gene correlations. We analyze a database of gene expression of Bacillus subtilis exposed to sub-lethal levels of 37 different antibiotics. Using unsupervised analysis (dendrogram) of the matrix of normalized gene-gene correlations, we identified the operons as they form distinct clusters of genes in the sorted correlation matrix. Applying dimension-reduction algorithm (Principal Component Analysis, PCA) to the matrices of normalized correlations reveals functional motifs. The genes are placed in a reduced 3-dimensional space of the three leading PCA eigen-vectors according to their corresponding eigen-values. We found that the organization of the genes in the reduced PCA space recovers motifs of the operon internal structure, such as the order of the genes along the genome, gene separation by non-coding segments, and translational start and end regions. In addition to the intra-operon structure, it is also possible to predict inter-operon relationships, operons sharing functional regulation factors, and more. In particular, we demonstrate the above in the context of the competence and sporulation pathways.

Conclusions/Significance

We demonstrated that by analyzing gene-gene correlation from gene-expression data it is possible to identify operons and to predict unknown internal structure of operons and gene-networks regulation.     

Analysis of the cellular functions of Escherichia coli operons and their conservation in Bacillus subtilis

The common assumption of operons as composed of genes that cooperate in a biological process is confirmed here by showing that Escherichia coli operons tend to be composed of genes that belong to the same general class of cellular function. Furthermore, the comparison between the genomic organization of E. coli and that of Bacillus subtilis shows that the genes that are homologous to genes that belong to experimentally characterized E. coli operons tend to cluster in neighboring regions of the genome. This tendency is greater for the subset of E. coli operons whose genes belong to a single functional class. These observations indicate strong evolutionary pressure that, translated into functional constraints, leads to the inclusion of many essential functions in conserved operons and clusters in these two distant species.     

Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria

Background

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.

Results

To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes.

Conclusions

We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins.     

Mannopinic acid and agropinic acid catabolism region of the octopine-type Ti plasmid pTi15955

Octopine-type Ti plasmids such as pTi15955, pTiA6 and pTiR10 direct the catabolism of at least eight compounds called opines that are released from crown gall tumours. Four of these compounds are denoted mannityl opines, each of which possesses a D -mannityl substituent on the nitrogen atom of either glutamate or glutamine. We have analysed a 20 kb region of the Ti plasmid pTi15955 that is required for the catabolism of two such opines, mannopinic acid and agropinic acid. A total of 12 genes in four operons were identified by DNA sequence analysis. Transposons Tn 5lacZ and MudK were used to mutagenize these genes and to create aga–lacZ and moa–lacZ translational fusions. The expression of all fusions was induced by agropinic acid and by mannopinic acid. One of these four operons encodes an agropinic acid permease, whereas a second one encodes a mannopinic acid permease. A third operon contains three genes encoding probable catabolic enzymes, two of which (AgaF and AgaG) are thought to convert agropinic acid to mannopinic acid, while the third (AgaE) probably converts mannopinic acid to mannose and glutamate. AgaE resembles a bacterial amino acid deaminase, whereas AgaF and AgaG resemble two bacterial proteins that together catabolize substituted hydantoins, whose chemical structure resembles that of agropinic acid. The remaining operon encoded the MoaR protein, a negative regulator of itself and of the other three operons.     

Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

Background  

A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target.