Lack of horizontal gene transfer of methicillin-resitance genetic determinants from PBP2a-positive, coagulase-negative Staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electric nerve stimulation (TENS),

Previous research shows that approximately half of the coagulase-negative staphylococci (CNS) isolated from patients in the intensive care unit (ICU) at Belfast City Hospital were resistant to methicillin. The presence of this relatively high proportion of methicillin-resistance genetic material gives rise to speculation that these organisms may act as potential reservoirs of methicillinresistance genetic material to methicillin-sensitive Staphylococcus aureus (MSSA). Mechanisms of horizontal gene transfer from PBP2a-positive CNS to MSSA, potentially transforming MSSA to MRSA, aided by electroporation-type activities such as transcutaneous electrical nerve stimulation (TENS), should be considered. Methicillin-resistant CNS (MR-CNS) isolates are collected over a two-month period from a variety of clinical specimen types, particularly wound swabs. The species of all isolates are confirmed, as well as their resistance to oxacillin by standard disc diffusion assays. In addition, MSSA isolates are collected over the same period and confirmed as PBP2a-negative. Electroporation experiments are designed to mimic the time/voltage combinations used commonly in the clinical application of TENS. No transformed MRSA were isolated and all viable S. aureus cells remained susceptible to oxacillin and PBP2a-negative. Experiments using MSSA pre-exposed to sublethal concentrations of oxacillin (0.25 µg/mL) showed no evidence of methicillin gene transfer and the generation of an MRSA. The study showed no evidence of horizontal transfer of methicillin resistance genetic material from MR-CNS to MSSA. These data support the belief that TENS and the associated time/voltage combinations used do not increase conjugational transposons or facilitate horizontal gene transfer from MR-CNS to MSSA.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

PHYLOGENETIC STUDY OF GEITLERINEMA AND MICROCYSTIS ( CYANOBACTERIA) USING PC-IGS AND 16S-23S ITS AS MARKERS: INVESTIGATION OF HORIZONTAL GENE TRANSFER

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Stochastic modeling of bacterial protein domains distribution to predict horizontal gene transfer

In questo elaborato, abbiamo tentato di modellizzare i processi che regolano la presenza dei domini proteici. I domini proteici studiati in questa tesi sono stati ottenuti dai genomi batterici disponibili nei data base pubblici (principalmente dal National Centre for Biotechnology Information: NCBI) tramite una procedura di simulazione computazionale. Ci siamo concentrati su organismi batterici in quanto in essi la presenza di geni trasmessi orizzontalmente, ossia che parte del materiale genetico non provenga dai genitori, e assodato che sia presente in una maggiore percentuale rispetto agli organismi più evoluti. Il modello usato si basa sui processi stocastici di nascita e morte, con l'aggiunta di un parametro di migrazione, usato anche nella descrizione dell'abbondanza relativa delle specie in ambito delle biodiversità ecologiche. Le relazioni tra i parametri, calcolati come migliori stime di una distribuzione binomiale negativa rinormalizzata e adattata agli istogrammi sperimentali, ci induce ad ipotizzare che le famiglie batteriche caratterizzate da un basso valore numerico del parametro di immigrazione abbiano contrastato questo deficit con un elevato valore del tasso di nascita. Al contrario, ipotizziamo che le famiglie con un tasso di nascita relativamente basso si siano adattate, e in conseguenza, mostrano un elevato valore del parametro di migrazione. Inoltre riteniamo che il parametro di migrazione sia direttamente proporzionale alla quantità di trasferimento genico orizzontale effettuato dalla famiglia batterica.

Horizontal gene transfer among genomes: The complexity hypothesis

Increasingly, studies of genes and genomes are indicating that considerable horizontal transfer has occurred between prokaryotes. Extensive horizontal transfer has occurred for operational genes (those involved in housekeeping), whereas informational genes (those involved in transcription, translation, and related processes) are seldomly horizontally transferred. Through phylogenetic analysis of six complete prokaryotic genomes and the identification of 312 sets of orthologous genes present in all six genomes, we tested two theories describing the temporal flow of horizontal transfer. We show that operational genes have been horizontally transferred continuously since the divergence of the prokaryotes, rather than having been exchanged in one, or a few, massive events that occurred early in the evolution of prokaryotes. In agreement with earlier studies, we found that differences in rates of evolution between operational and informational genes are minimal, suggesting that factors other than rate of evolution are responsible for the observed differences in horizontal transfer. We propose that a major factor in the more frequent horizontal transfer of operational genes is that informational genes are typically members of large, complex systems, whereas operational genes are not, thereby making horizontal transfer of informational gene products less probable (the complexity hypothesis).

Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35s-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

Adaptation of yeasts to fructose rich environments: a role for horizontal gene transfer of a fructose transporter

Abstract of the poster presented 33rd Small Meeting on Yeast Transport and Energetics, 21-24 July 2015, Lisbon, Portugal.

Replacement of the arginine biosynthesis operon in Xanthomonadales by ateral gene transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the gamma-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.