Activation of r20-dependent recombination and horizontal gene transfer in Mycoplasma genitalium

In the human pathogen Mycoplasma genitalium, homologous recombination is under the control of r, an alternative sigma factor that boosts the generation of genetic and antigenic diversity in the population. Under laboratory growth conditions, r activation is rare and the factors governing its intermittent activity are unknown. Two r-regulated genes, rrlA and rrlB, showed to be important for recombination of homologous DNA sequences in this bacterium. Herein, we demonstrate that rrlA and rrlB code for two small proteins that participate in a feed-forward loop essential for r function. In addition, we identify novel genes regulated by r and show that several non-coding regions, which function as a reservoir for the generation of antigenic diversity, are also activated by this alternative sigma factor. Finally, we reveal that M. genitalium cells can transfer DNA horizontally by a novel mechanism that requires RecA and is facilitated by r overexpression. This DNA transfer system is arguably fundamental for persistence of M. genitalium within the host since it could facilitate a rapid dissemination of successful antigenic variants within the population. Overall, these findings impose a novel conception of genome evolution, genetic variation and survival of M. genitalium within the hos

All-in-one construct for genome engineering using Cre-lox technology

Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications

Multifunctional Proteins : Involvement in Human Diseases and Targets of Current Drugs

Multifunctionality or multitasking is the capability of some proteins to execute two or more biochemical functions. The objective of this work is to explore the relationship between multifunctional proteins, human diseases and drug targeting. The analysis of the proportion of multitasking proteins from the MultitaskProtDB-II database shows that 78% of the proteins analyzed are involved in human diseases. This percentage is much higher than the 17.9% found in human proteins in general. A similar analysis using drug target databases shows that 48% of these analyzed human multitasking proteins are targets of current drugs, while only 9.8% of the human proteins present in UniProt are specified as drug targets. In almost 50% of these proteins, both the canonical and moonlighting functions are related to the molecular basis of the disease. A procedure to identify multifunctional proteins from disease databases and a method to structurally map the canonical and moonlighting functions of the protein have also been proposed here. Both of the previous percentages suggest that multitasking is not a rare phenomenon in proteins causing human diseases, and that their detailed study might explain some collateral drug effects

Transcriptional response to metal starvation in the emerging pathogen Mycoplasma genitalium is mediated by Fur-dependent and -independent regulatory pathways

Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur- and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition

A hypothesis explaining why so many pathogen virulence proteins are moonlighting proteins

Moonlighting or multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Proteins that belong to key ancestral functions and metabolic pathways such as primary metabolism typically exhibit moonlighting phenomenon. We have collected 698 moonlighting proteins in MultitaskProtDB-II database. A survey shows that 25% of the proteins of the database correspond to moonlighting functions related to pathogens virulence activity. Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? Our hypothesis is that these proteins present a high conservation between the pathogen protein and the host counterparts. Therefore, the host immune system will not elicit protective antibodies against pathogen proteins. The fact of sharing epitopes with host proteins (known as epitope mimicry) might be the cause of autoimmune diseases. Although many pathogen proteins can be antigenic, only a few of them would elicit a protective immune response. This would also explain the lack of successful vaccines based in these conserved moonlighting proteins. This review looks at why so many pathogen virulence proteins are from the primary metabolism and are conserved between pathogen and host

MultitaskProtDB-II : an update of a database of multitasking/moonlighting proteins

Multitasking, or moonlighting, is the capability of some proteins to execute two or more biological functions. MultitaskProtDB-II is a database of multifunctional proteins that has been updated. In the previous version, the information contained was: NCBI and UniProt accession numbers, canonical and additional biological functions, organism, monomeric/oligomeric states, PDB codes and bibliographic references. In the present update, the number of entries has been increased from 288 to 694 moonlighting proteins. MultitaskProtDB-II is continually being curated and updated. The new database also contains the following information: GO descriptors for the canonical and moonlighting functions, three-dimensional structure (for those proteins lacking PDB structure, a model was made using Itasser and Phyre), the involvement of the proteins in human diseases (78% of human moonlighting proteins) and whether the protein is a target of a current drug (48% of human moonlighting proteins). These numbers highlight the importance of these proteins for the analysis and explanation of human diseases and target-directed drug design. Moreover, 25% of the proteins of the database are involved in virulence of pathogenic microorganisms, largely in the mechanism of adhesion to the host. This highlights their importance for the mechanism of microorganism infection and vaccine design. MultitaskProtDB-II is available at http://wallace.uab.es/multitaskII

P110 and P140 cytadherence-related proteins are negative effectors of terminal organelle duplication in Mycoplasma genitalium

Background: The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication. - Methodology/Principal Findings: in this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3' end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas. - Conclusions/Significance: our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism

All-in-one construct for genome engineering using Cre-lox technology

Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications

Gene ontology function prediction in Mollicutes using protein-protein association networks

Many complex systems can be represented and analysed as networks. The recent availability of large-scale datasets, has made it possible to elucidate some of the organisational principles and rules that govern their function, robustness and evolution. However, one of the main limitations in using protein-protein interactions for function prediction is the availability of interaction data, especially for Mollicutes. If we could harness predicted interactions, such as those from a Protein-Protein Association Networks (PPAN), combining several protein-protein network function-inference methods with semantic similarity calculations, the use of protein-protein interactions for functional inference in this species would become more potentially useful. In this work we show that using PPAN data combined with other approximations, such as functional module detection, orthology exploitation methods and Gene Ontology (GO)-based information measures helps to predict protein function in Mycoplasma genitalium. To our knowledge, the proposed method is the first that combines functional module detection among species, exploiting an orthology procedure and using information theory-based GO semantic similarity in PPAN of the Mycoplasma species. The results of an evaluation show a higher recall than previously reported methods that focused on only one organism network

Estudio de la expresión génica y la división celular en Mycoplasma genitalium

Descripció del recurs: el 27 de juny de 2011Los micoplasmas son bacterias sin pared celular que pertenecen a la clase Mollicutes. Estos microorganismos se caracterizan por su pequeño tamaño y por tener un genoma reducido con un bajo porcentaje de C+G. Muchas especies de mycoplasma actúan como parásitos y patógenos de un amplio rango de huéspedes, causando enfermedades comunes en humanos. Por ejemplo, Mycoplasma genitalium, objeto de esta tesis, es el causante de la uretritis no gonocócica. La secuenciación del genoma de algunos micoplasmas ha favorecido el conocimiento de la fisiología y genética de estos microorganismos, pero algunos mecanismos como la regulación de la expresión génica y la división celular siguen siendo aún un misterio por desvelar. M. genitalium, con tan sólo 525 genes, es considerado un modelo de célula mínima. Posee el genoma más pequeño de entre todas las bacterias que se pueden cultivar axénicamente, siendo así un candidato ideal para el estudio de los procesos biológicos con el mínimo número de genes implicados. El trabajo presentado en esta tesis se divide en tres capítulos independientes. En el primero se estudia la regulación de la expresión génica en M. genitalium y M. pneumoniae. El trabajo con M. pneumoniae fue llevado a cabo durante una estancia en el laboratorio del Prof. J. Stülke (Göttingen, Alemania). Este primer trabajo dio lugar a un artículo publicado en la revista Microbiology. La obtención de un mutante por transposición de M. genitalium que deleccionaba el gen ftsZ, descrito como esencial para la división celular en la mayor parte de bacterias, derivó en el segundo capítulo de esta tesis. En este segundo trabajo se investiga la división celular en micoplasma en ausencia de este gen. Este estudio ha sido publicado recientemente en la revista Molecular Microbiology. Por último, con el objetivo de profundizar en el tema de la división celular en micoplasma, en el tercer capítulo se analiza la funcionalidad del gen mraZ, que junto con mraW, mg223 y ftsZ conforma el operón de división celular de M. genitalium.Mycoplasmas are the smallest and simplest free-living microorganisms. They are members of the Mollicutes class which is characterized by the absence of cell wall and by having genomes with a low G+C content. Despite this apparent simplicity, some mycoplasma species are parasites and pathogens of a wide range of hosts. For instance, Mycoplasma genitalium, which is the object of this thesis, is the agent of non-gonococcal and non-chlamydial urethritis. Genome sequencing has advanced the knowledge concerning the physiology and genetics of these microorganisms, but some mechanisms such as the regulation of gene expression and the cell division remain unclear. With only 525 genes M. genitalium is considered a model of minimal cell, since it has the smallest genome of any microorganism that can be grown in a pure or axenic culture. It is considered a perfect candidate to study biological processes with the minimal set of genes. This thesis is divided into three independent chapters. In the first one we study the regulation of gene expression in M. genitalium and M. pneumoniae. This work was published in 2007 in the journal "Microbiology". A minitransposon insertion was found in the ftsZ gene of M. genitalium, indicating that this gene was not essential for cell division in this microorganism. In the second chapter of this thesis we investigate the cell division process in M. genitalium in the absence of ftsZ. This work was recently published in the journal "Molecular Microbiology". Finally, to gain insight into the cell division process in mycoplasma, in the third chapter we analyze the function of the mraZ gene, which together with mraW, mg223 and ftsZ comprise the cell division operon of M. genitalium