Saturday, January 31, 2009

Qui gladio ferit, gladio perit

Bacterial conjugation-based antimicrobial agents.
Marcin Filutowicz, Richard Burgess, Richard L. Gamelli, Jack A. Heinemann, Brigitta Kurenbach, Sheryl A. Rakowski, Ravi Shankar
Plasmid 60 (2008) 38–44

Horizontal gene transfer is an essential mechanism in the adaptive evolution of bacteria. In genomic era it is more evident that the antibiotic resistance genes are spread across microbial population. It is also known that these genes are often located on mobile genetic elements, of which conjugative plasmids represent a major group and are found in nearly all Prokaryotes. Broad host range plasmids and conjugation are the main ways of spreading antibiotic resistance through bacterial population. But “He who lives by the sword shall die by the sword”, so could we take advantage of conjugation and use it to kill pathogenic bacteria.
In this short review paper authors summarize the work on so called bacterial conjugation-based technologies (BCBT). These technologies exploit plasmid biology for combating the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumulated work of many scientists dating back to the discoveries of conjugation and plasmids themselves.
It is easy to imagine how it works. Genetic information carried by plasmid DNA is expressed in the recipient cells upon conjugation. So if plasmids carry instruction for destruction of the host cells it is executed. Authors present 3different ways to kill the host cell by plasmid.

The first approach is very simple. It uses so called runaway plasmid which can replicate without any control in a host cell. Replication of plasmid DNA acts like a trap to capture all of the cell’s available replication machinery to the exclusion of chromosomal replication.

The second is production of plasmid- or chromosome-encoded bacteriocins. In almost all instances, cells producing a bacteriocin also produce a bacteriocin-specific antidote, typically a peptide or RNA. For BCBT purpose, an anti-kill antidote, which can neutralize the expression of a plasmid-encoded antimicrobial agent, can be integrated into the chromosome of the donor bacteria. Susceptible recipients are killed after plasmid transfer from the protected donor cells.

In the third approach, a donor might be rendered insensitive to a killer plasmid by using a tightly regulatable promoter-operator system in which the expression of a lethal bacteriocin gene is prevented by a repressor made only in the donor cell. An engineered example of a plasmid with multiple toxins that are independently regulated has been built and employed in the proof-of-concept experiments which are described in the paper.

The BCBT has been successfully used by ConjuGon Inc. (Madison, WI), and the Loyola University Medical Center’s Burn and Shock Trauma Institute (Maywood, IL), in eradicating Acinetobacter baumannii in vitro and in an in vivo murine burn sepsis model. A. baumannii is a Gram-negative opportunistic human pathogen that is found in soil and water and is easily transmitted in health care settings. Wounds such as burns are routinely treated with topical antibiotics at high enough doses to achieve therapeutic concentration; however, such antibiotic treatment is compromised if the wound is infected with multidrug- resistant bacterial strains. Many clinically-isolated strains of A. baumannii are pan resistant (resistant to all antibiotics) and the incidence of nosocomial infections caused by such strains is increasing in critically injured and immunocompromised patients who are hospitalized for prolonged periods. So BCBT could be extremely useful in such cases.
The other target for BCBT is to kill pathogenic bacteria which are living inside the host cells. These bacteria establish themselves in the intracellular milieu of their host, thereby evading administered antibiotics as well as the host’s immune system. To target those pathogens the intra- or inter-species conjugation can be used. In that case a non pathogenic strain (such as Salmonella) acts a donor of killer-plasmid. In the case of Mycobaterium tuberculosis and M. avium infections bacteriophages were used instead of plasmid to reduce number of pathogens.

There are also some disadvantages of this technology. The efficiency of killing pathogenic bacteria depends first on plasmid transfer efficiency, and second, on plasmid killing properties itself. Thus it is important to increase the ability of plasmid to be transfer to the specific target host. It is also good to find highly efficient killing system. But even with high efficient conjugation and killing system it seems to be very unlikely to eliminate all susceptible bacteria in the environment, because they still have some “defense” systems like restriction systems to protect. On the other hand the commercial antibiotics also do not kill all susceptible bacteria. But the decreased number of pathogenic cells allows immunological system to finish the job.

Another thing is the use of bacterial strains containing modified genetic information and “releasing” them to the “environment”. Authors present an assortment of applications of live bacteria approved by U.S. government agencies for use or further study.

To summarize, in some cases the BCBT could be an alternative method of dealing with bacterial infections and as a new technology can be developed in all possible ways...

dr Jaroslaw E. Krol

Friday, January 23, 2009

Plasmid gene content analysis

Plasmid gene content analysis

Shared gene content patterns, also called phylogenetic profiles, have been used to build phylogenetic trees (Snel et al. 1999), to predict protein function (Pellegrini et al. 1999), and to reconstruct gene content of ancestral species (Kunin et al. 2003) for prokaryotic genomes. The phylogenetic reconstruction based on gene content is useful particularly for mobile genetic elements such as phages and plasmids where universally shared homologous sequences, a prerequisite for phylogenetic analyses, are not always available. Recently, the gene content analysis has been applied to phages (Lima-Mendez et al. 2008). Most recently, Brilli et al. (2008) applied this to plasmids from Enterobacteriaceae family of gamma-Proteobacteria including Escherichia, Salmonella and Shigella genera. The authors stated that 'most of plasmids does not form tight clusters coherent with the taxonomic status of their respective host species (E. coli, Salmonella or Shigella). This finding suggest a complex evolutionary history of such plasmid replicons with massive horizontal transfer and gene rearrangements.'

In contrast to other researchers, Brilli et al. (2008) did not discuss the performance of the phylogenetic profiling methods for plasmids. For example, Snel et al. (1999) demonstrated the correlation of prokarytic phylogeny based on gene content with that based on sequence similarity of 16S rRNA. Also, Lima-Mendez et al. (2008) clustered phage genes based on their phylogenetic profiles to define evolutionary cohesive modules, and showed that in temperate phages evolutionary modules correspond better to functional modules, whereas in virulent phages they span several functional categories. This suggests that the phylogenetic profiling does not always work well at predicting protein function in phages.

This has inspired us to validate the performance of the gene content analysis with the set of genes shared as orthologs by all members of an evolutionarily coherent plasmid group (such as IncFI, IncFII, IncI1, IncN, IncP-1, and IncW), and by focusing on functionally linked proteins such as those involved in the replication, maintenance, and conjugative transfer of plasmids.

Brilli M, Mengoni A, Fondi M, Bazzicalupo M, Lio P, Fani R. BMC Bioinformatics. (2008) 9(1):551. Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network.

Snel B, Bork P, Huynen MA. Nat Genet. (1999) 21(1):108-10. Genome phylogeny based on gene content.

Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. Proc Natl Acad Sci U S A. (1999) 96:4285-8. Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.

Kunin V, Ouzounis CA. Bioinformatics. (2003) 19:1412-6. GeneTRACE-reconstruction of gene content of ancestral species.

Lima-Mendez G, Van Helden J, Toussaint A, Leplae R. Mol Biol Evol. (2008) 25:762-77. Reticulate representation of evolutionary and functional relationships between phage genomes.

Dr. Haruo Suzuki
University of Idaho

Thursday, January 15, 2009

Changing each other’s lives and transcription

One of the challenges of biology is that life does not function as a vacuum, but rather constantly influences and is influenced by other organisms and the environment. Therefore, in order to unravel some of the mysteries of the functions and evolution of living things, one must examine not only the organism itself, but also those things that may be influencing it. For bacteria, horizontal gene transfer (HGT) is a key process by which a bacterium is influenced by its environment and the genetic organization of neighboring bacteria. Through HGT a bacterium can acquire plasmids that confer upon the bacterial host new phenotypic traits. In the case of plasmid pCAR1, hosts receive the genetic information necessary to degrade carbazole and therefore use it as a carbon source. However, the gain or loss of a plasmid and its inherent phenotypic properties are not the extent of how plasmids and bacteria influence one another. As the authors of this paper pointed out, the host’s chromosomal transcriptome can be influenced by the presence of a plasmid and that, conversely, transcription of plasmid backbone and accessory genes are affected by the chromosome of the host in which the plasmid finds itself.

Past research by these authors showed that pCAR1 could successfully transfer to and function in Pseudomonas putida KT2440 from its original host, Pseudomonas resinovorans CA10. They also found that the introduction of the pCAR1 affected chromosomal transcription. In this study, they pursued the question of if and how plasmid transcription of pCAR1 is affected by different host chromosomes, again using P. putida KT2440 and P. resinovorans CA10. To do this they used a microarray to map and quantify PCAR1 transcripts in the two hosts when grown on succinate or carbazole as the only available carbon source. They also verified their results from the microarrays through real-time PCR. Through these two methods, the authors verified that growth on carbazole induced the catabolic operons antA and antR to the same extent, regardless of the host. However, other genes, such as the car operons, were expressed at significantly different levels depending on which host the plasmid was acting in.

This article adds to the growing body of evidence that the expression of genomes are not static bodies with occasional changes due only to mistakes made from one generation of cells to another, but is rather a dynamic entity that is, like the organism itself, influenced by its immediate environment at any given time.

Primary article:

Miyakoshi M, H Nishida, M Shintani, H Yamane, H Nojiri. (2009). High-resolution mapping of plasmid transcriptomes in different host bacteria. BMC Genomics, 10:12.

Additional reading:

Frost LS, Leplae R, Summers AO, Toussaint A. (2005). Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol 3:722-32.

Harr B, Schlötterer C. (2006). Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome. BMC Microbiol 6:80.

Miyakoshi M, Sintani M, Terabayashi T, Kai S, Yamane H, Nojiri H. (2007). Transcriptome analysis of Pseudomonas putida KT2440 harboring the completely sequenced IncP-7 plasmid pCAR1. Bacteriol. 189: 6849-60.

Ramos JL, Marqués S, Timmis KN. (1997). Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rec Microbiol. 51: 341-72.

Thomas CM. (2006). Transcription regulatory circuits in bacterial plasmids. Biochem Soc Trans. 34:1072-4.

Julie M. Hughes
University of Idaho

Saturday, January 10, 2009

Cupriavidus metallidurans: evolution of a metal-resistant bacterium

Torsten von Rozycki Æ Dietrich H. Nies

Cupriavidus metallidurans CH34 is a gram-negative bacterium that is frequently found in soils and sediments with a high content of heavy metals and is therefore resistant to multiple heavy metals such as Zn, Cd, Co, Pb, Cu, Hg, Ni and Cr. C. metallidurans CH34 has a large mega-plasmid and two large plasmids (pMOL28 and pMOL30) that have low copy numbers and can be maintained in the cell even without selective pressure. Resistance to these metals is mediated by transmembrane protein complexes, which export cations from the cytoplasm to the exterior of the cell. The main question this work is attempting to answer is: “ How did C. metallidurans CH34 acquire so many metal resistance genes? ”

To answer this question, the authors decided to analyze the genomes of seven bacteria to study the occurrence of orthologous and paralogous proteins coding for metal resistance. All seven bacteria belong to the β-proteobacterial family Burkholderiaceae of the order Burkholderiales. These include the hydrogen oxidizing C. eutrophus strain H16 (Pohlmann et al. 2006) and the xenobiotic degrader C. eutrophus JMP134 along with 2 phytopathogenic bacteria Ralstonia solanacearum strain GMI1000 (Salanoubat et al. 2002) and strain UW551. The last two organisms that were included were Burkholderia xenovorans strain LB400 and Burkholderi cepacia strain AMMD and were taxonomically distinct from Ralstonia and Cupriavidus. For this analysis, a standardized database for transporter proteins TCDB ( was used as a reference. The latest releases of protein sequences of all seven strains were obtained from JGI and NCBI. These were then blasted against the TCDB database (Busch and Saier 2002). A total of seven transporter protein classes (channels/pore, electrochemical potential-driven
transporters, primary active transporters, PTS-group translocators, transport electron carriers, accessory factors involved in transport, incompletely characterized transport
systems) were found in all of the seven genomes. These transporter proteins differed from each other based on the method of transport and also on the mechanism of energy utilization (Saier 2000; Saier et al. 2006). It was also seen that the number of transporter proteins per Mb was similar in all of the strains (and most of the plasmids) analyzed. Thus, the authors conclude that metal resistance in C. metallidurans is not due to a higher number of transport proteins.

In the next step the authors analyzed the paralogs in all seven strains. Paralogs arise by gene duplication in an organism. A high percentage of protein coding paralogs were found on the plasmids of CH34 (34%), H16 (31%) and JMP134 (21%). Moreover, half of the transport proteins found on plasmids of CH34 were paralogs. For instance, the plasmid pMOL30 had a higher percentage of paralogous proteins than any of the other plasmids or chromosomes. The authors surmise that evolution of CH34 has been due to the duplication of transport proteins on its plasmids. The same mechanism may have been responsible for the evolution of the strains H16 and JMP134. Orthologs were investigated next. Here also, pMOL30 exhibited an unusually low percentage (17%) of orthologous proteins. This fact along with the high number of paralogs on plasmid pMOL30 may indicate that gene duplication and horizontal gene transfer played important roles in the evolution of this plasmid.

A total of 700 transport proteins were common among the three Cupriavidus strains. The transport proteins of CH34 could be assigned to twenty protein families based on the classification of the TCDB database. The twenty protein families had orthologs in all strains, however; some protein families were present more than once in CH34. Examples include the Mot/Exb complex components that energize active transport across the outer membrane, ABC transport systems, and metal inorganic transport (MIT) systems, RND, MFP and OMF protein families, P-type ATPases, proteins of the major facilitator superfamily (MFS), and components of the type III (TTS) and the type IV (TFS) secretion systems. Since all of the above proteins export cations, this shows that CH34 has twice as many of these proteins as the other six strains.
Next, the number of protein families involved in the transport of transition metals such as CDF, MerTP, MFP, MIT, NiCoT, OMF, OMR, P-type ATPase, CHR, HME/RND, and ZIP protein families was studied in the seven strains. The authors found that CH34 had a much higher number of the above protein families than the other bacterial strains (i.e., 83 compared to between 44 and 69). When genome size was taken into consideration it was shown that CH34 had 12 transition metal transport proteins per Mb while all the other six bacteria had 6–8 such proteins per Mb. Thus CH34 seems to have evolved its metal resistance by horizontal gene transfer and gene duplication.
RND proteins are a superfamily of proteins that are part of multi subunit protein complexes involved in efflux reactions (Tseng et al. 1999). A subgroup of this family called the HME-RND proteins are involved in the efflux of metals. CH34 has twelve HME-RND operons (Nies 2003), while the other six bacteria have fewer than twelve. This means that the number of operons has steadily increased in CH34 probably by horizontal gene transfer. Three of these twelve operons were vigorously expressed in CH34 and code for the following: the chromosomal copper/silver HME4-RND system, cnr for cobalt/nickel resistance on plasmid pMOL28, and czc for cobalt/zinc/cadmium resistance on plasmid pMOL30. The cobalt/zinc/cadmium resistance operon is czcICBA (Nies 2003) which is found not only on pMOL30, but also on CH34 chromosome 2 and has homologs on chromosome 2 of both C. eutrophus strains. Thus the authors conclude that all three strains might have inherited a czcICBA-like operon on chromosome 2 from an ancestral Cupriavidus strain and in CH34 this operon was duplicated onto plasmid pMOL30. Another operon, czcDRSE, (Große et al. 1999, 2004) is located downstream of the czcICBA operon on pMOL30 and encodes the CDF protein CzcD which transports divalent cations. The authors suggest that this operon was probably assembled by the horizontal transfer of czcD and regulatory genes czcRS along with the duplication of the copH gene (from the copper resistance cluster on pMOL30) to form czcE. Since czcE binds copper, it may form a link between the czcDRSE and czcICBA operons. Similarly, nickel/cobalt resistance is encoded on pMOL28 by the cnrYXHCBA operon (Liesegang et al. 1993), which has no homologs on any of the other bacterial strains. This operon, too, may have been acquired by horizontal gene transfer. P-type ATPases form a family of membrane-bound primary transport systems (Fagan and Saier 1994). Strain CH34 contains a high number of 13 predicted P-type ATPases. The other two Cupriavidus strains 7 or 8 orthologs including Ca2+ and Zn2+/Cd2+/Pb2+ exporting enzymes. Cupriavidus metallidurans contains four CHR proteins that export chromate from the cytoplasm (Nies 2003; Nies et al. 1998). This too could have been a result of gene duplication after speciation from the ancestral Cupriavidus strain. During speciation of C. metallidurans CH34 two MerT proteins duplicated into four, yielding three active mercury-detoxification systems. The authors summarize by saying that “ the ancestral Cupriavidus strain might have been a facultatively hydrogen-oxidizing, moderately metal-resistant degrader of aromatic compounds and organic acids rather than a dweller on sugars .” This strain evolved by the acquisition of plasmids such as those that carry hydrogen-oxidizing genes, metal resistance genes such as nickel, cobalt, chromate, and mercury, as well as genes coding for degradation of organic compounds such as 2,4-D. CH34 in particular probably evolved by a combination of horizontal gene transfer and gene duplication events along with rearrangements on Pmol30 which lead to adaptation of this strain to a wide range of metals.
It is now a known fact that horizontal gene transfer plays a crucial role in prokaryotic evolution. Studies such as these are important since they provide evidence of the role of horizontal gene transfer in the evolution of a complex strain such as CH34. Detailed analysis of each operon on the CH34 strain made it possible to trace its origin from the ancestral strain.


Pohlmann A, Fricke WF, Reinecke F et al (2006) Genome sequence of the bioplastic-producing ‘‘Knallgas’’ bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–

Salanoubat M, Genin S, Artiguenave F et al (2002) Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497–502.

Busch W, Saier MHJ (2002) The transporter classification (TC) system. Crit Rev Biochem Mol Biol 37:287–337.

Saier MHJ (2000) A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64:354–411

Saier MHJ, Tran CV, Barabote RD (2006) TCDB: the transporter classification database for membrane transport protein analyses and information. Nucleic Acids Res

Tseng T-T, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A et al (1999) The RND superfamily: an ancient, ubiquitous and diverse family that includes human disease
and development proteins. J Mol Microbiol Biotechnol 1:107–125.

Nies DH (2003) Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 27:313–339.

Große C, Grass G, Anton A, Franke S, Navarrete Santos A, Lawley B et al (1999) Transcriptional organization of the czc heavy metal homoeostasis determinant from Alcaligenes eutrophus. J Bacteriol 181:2385–2393

Große C, Anton A, Hoffmann T, Franke S, Schleuder G, Nies DH (2004) Identification of a regulatory pathway that controls the heavy metal resistance system Czc via promoter
czcNp in Ralstonia metallidurans. Arch Microbiol 182:109–118

Liesegang H, Lemke K, Siddiqui RA, Schlegel H-G (1993) Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes
eutrophus CH34. J Bacteriol 175:767–778

Fagan MJ, Saier MH Jr (1994) P-type ATPases of eukaryotes and bacteria: sequence comparisons and construction of phylogenetic trees. J Mol Evol 38:57–99.

Nies DH, Brown N (1998) Two-component systems in the regulation of heavy metal resistance. In: Silver S, Walden W (eds) Metal ions in gene regulation. Chapman Hall,
London, pp 77–103

Diya Sen
University of Idaho