Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

De Bast M. S., Mine N. and Van Melderen L. (2008) J. Bacteriol. 190:4603-4609

Plasmids are known as carriers of pathogenic determinants and antibiotic resistance genes that help bacteria survive in specific circumstances. However, if the circumstances changed, the bacteria would no longer need such genes, so that the plasmid would become just a burden for bacteria. As a result, plasmid-free bacterial cells would increase their number more rapidly than plasmid-carrying cells. Most of naturally occurring plasmids have special systems to prevent such an event from happening. One of the systems is the Toxin-Antitoxin (TA) system, in which one gene encodes a stable toxin protein and the other gene encodes an unstable antitoxin protein that counteracts the toxin activity. If the plasmid carrying the TA system was lost from a cell, the cell would be immediately killed or damaged by the more stable toxins which persist in the cell; this phenomenon is called postsegregational killing [PSK]. Gene pairs comprising TA systems are called addiction modules. Addiction modules were originally discovered on a plasmid (Hiraga et al., 1986), but recently they have also been discovered on chromosomes (reviewed by Kobayashi I., 2004). Here, we have a question: What is the biological function of chromosomally-located addiction modules?

The first hypothesis proposed is the so-called programmed cell death (PCD) hypothesis: the addiction module induces cell death under stress conditions and the dead cells release nutrients for other cells to remain viable (Aizenman et al., 1996). Recently, this hypothesis has been shown to be unlikely by several research groups (Tsilibaris et al., 2007; Szekeres et al., 2007; Dudde et al., 2007; Pedersen et. al., 2002). The second and more reasonable hypothesis is that addiction modules contribute to stabilize a genome: the toxin reduces the number of bacteria that have lost the chromosomal DNA segment containing the addiction modules, which ensures that the DNA in the region of the addiction module is maintained in the bacterial population (Szekeres et al., 2007).

In this paper, the authors propose a third theory: the "anti-addiction module" hypothesis. In this hypothesis, addiction modules on a chromosome protect bacteria against PSK induced by orthologous addiction modules on a plasmid, which confers selective advantage on a host bacterium under PSK conditions.

To test the anti-addiction module hypothesis, the authors used the CcdB(F)/CcdA(F) TA system of F-plasmid (Hiraga et al., 1986) and its homologous system [CcdB(Ech)/CcdA(Ech) TA system] found in the Escherichia chrysanthemi chromosome; CcdB(F) toxin is 61% identical to CcdB(Ech) while CcdA(F) antitoxin is 65% identical to CcdA(Ech). In this article, the authors first showed that the ccdB(Ech) and ccdA(Ech) genes indeed act as toxin and antitoxin genes in E. coli MG1655 where the ccdB(F)/ccdA(F) homologous genes are absent. However, unlike F-plasmid's ccdB(F)-ccdA(F) gene pair, the ccdB(Ech)-ccdA(Ech) gene pair could not mediate PSK when it was cloned on a plasmid. This suggests that the two homologous TA systems have evolved for different purposes. The authors also showed that CcdA(Ech) can antagonize CcdB(F) toxic activity as efficiently as CcdA(F) can. Furthermore, they showed that the E. coli MG1655 derivative that carries the ccdB(Ech)-ccdA(Ech) gene pair on its chromosome (designated MG1655ccdEch) are more viable than the original MG1655 after the induction of PSK mediated by F-plasmid's ccdB(F)-ccdA(F) gene pair on a plasmid. The following competition assays between MG1655 and MG1655ccdEch in PSK conditions indicated that MG1655ccdEch has a 25% fitness advantage over MG1655. Therefore, all experiments performed in this article support anti-addiction module hypothesis. A related idea was also proposed by Takahashi et al. (2002), using a restriction-modification TA system, in which restriction enzymes act as toxins and modification methyltransferases act as antitoxins. They showed that dcm methyltransferase gene located on the E. coli chromosome protected cells against PSK mediated by a restriction enzyme and DNA modification gene pair on a plasmid.

If the anti-addiction module hypothesis is valid, there would be few cases in nature that counteracting addiction modules are found on both plasmid and chromosome in the same cell, because PSK does not happen in such a situation and consequently there would be no advantage for plasmids to carry the addiction module. However, in the genome of E. coli O157:H7, two homologous ccdB-ccdA gene pairs exist. One ccdB-ccdA gene pair is located on plasmid pO157 and the other is located on the chromosome. The ccd genes of pO157 are identical to F-plasmid's counterparts. Chromosomal ccdB and ccdA gene products, CcdB(O157) and CcdA(O157), are 35% and 30% identical to CcdB(F) and CcdA(F), respectively. As we can expect, the chromosomal ccdA-ccdB gene pair of O157:H7 does not counteract CcdB(F) toxicity and O157:H7 is susceptible to PSK mediated by the CcdB(F)/CcdA(F) TA system (Wibaux et al., 2007).

The integration of addiction modules into the chromosome can protect bacteria from plasmids that may have a high cost under some conditions. However, as the authors state in this article, that in turn drives the evolution of plasmid TA systems so as not to be counteracted by chromosomal TA systems. It thus appears to me that the primal role of chromosomal TA systems is maintaining the integrity of chromosomes in bacterial populations and the secondary role may be protecting bacteria against PSK mediated by invader DNA elements such as phages and plasmids. Do you have another hypothesis? If so, please let me know.


References:
Aizenman E, Engelberg-Kulka H, Glaser G. (1996) An Escherichia coli chromosomal "addiction module" regulated by guanosine 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl. Acad. Sci. 93:6059-6063.

Budde PP, Davis BM, Yuan J, Waldor MK. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J Bacteriol. 189:491-500

Hiraga S, Jaffé A, Ogura T, Mori H, Takahashi H. (1986) F plasmid ccd mechanism in Escherichia coli. J. Bacteriol. 166:100-104.

Kobayashi I. (2004) Genetic Addition: a principle of Gene symbiosis in a Genome, in Plasmid Biology (Funnell B. E. and Phillips G. J. eds), pp.105-144 ASM press, Washigton D.C.

Pederson K, Christensen SK, and Gerdes K (2002) Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol. Microbiol. 45:501-510.

Szekeres S, Dauti M, Wilde C, Mazel D, Rowe-Magnus DA. (2007) Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63:1588-1605.

Tsilibaris V, Maenhaut-Michel G, Mine N, Van Melderen L (2007) What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome? J. Bacteriol. 189:6101-6108.

Takahashi N, Naito Y, Handa N, Kobayashi I. (2002) A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. J. Bacteriol. 184:6100-6108.

posted by Hirokazu Yano, University of Idaho

Who will win this war…….?

High in vitro antimicrobial activity of synthetic antimicrobial
peptidomimetics against staphylococcal biofilms.

Kristina Flemming, Claus Klingenberg, Jorun Pauline Cavanagh, Merethe Sletteng, Wenche Stensen, John Sigurd Svendsen and Trond Flægstad

Journal of Antimicrobial Chemotherapy (2009) 63, 136–145

It is more than 80 years when the first antibiotic was discovered by Sir Alexander Fleming (September 28, 1928). Since the mid 40’s antibiotics have been widely used to prevent bacterial outbreaks in humans, but they also play a role as growth promoting agents in agriculture. The years of positive selection pressure have caused the global spread of antibiotic resistance in the microbial population. Special threats are bacteria that form a biofilm. Biofilm is defined as microbial-derived sessile communities attached to a surface and embedded in a self-produced polymeric matrix. Bacteria in biofilms are usually less susceptible to antimicrobial agents than rapidly growing planktonic cells. There are several hypotheses to explain the strong antimicrobial tolerance of biofilm cells such as the limitation of agent penetration, the existence of dormant cells, phenotypic variations, a quorum sensing system, and multidrug efflux pumps. So there is always need to develop new effective antimicrobial agents that can kill bacteria.

Cationic antimicrobial peptides (CAPs) are widespread in nature and play an important role as part of innate immunity. In general, CAPs are fairly large molecules that carry a net positive charge and contain ~50% hydrophobic residues. Their mode of action involves binding to negatively charged structural molecules on the microbial membrane. Once bound, CAPs form pores that increase the cell membrane permeability and ultimately lead to cell lysis. There is also evidence for other antimicrobial mechanisms such as interaction with intracellular targets or activation of autolytic enzymes in the bacteria, or induction of the immune response in the host. CAPs have a broad spectrum of antimicrobial activity and development of resistance is rare. Unfortunately, CAPs are difficult and expensive to produce in large quantities and are usually sensitive to protease digestion. Modifications of CAPs have resulted in the development of extremely short synthetic antimicrobial peptidomimetics, also called SAMPs. SAMPs mimic the effect of CAPs, but have improved pharmacokinetic properties and are thus a promising new group of antimicrobial substances.

In this study the authors investigated the antimicrobial activity of clinically relevant antibiotics like linezolid, tetracycline, rifampicin and vancomycinand, and newly designed SAMPs against biofilms of three different staphylococcal species (six strains). They also evaluate a simple screening method to quantify the metabolic activity of biofilms before and after the biofilm had been subjected to treatment with antimicrobial agents.

Two methods were used for quantify the biofilm metabolic activity. The first method used Alamar Blue (AB) to measure metabolic activity. AB is a redox indicator that both fluoresces and changes color in response to chemical reduction that can be measured by monitoring absorbances at 570 and 600nm. The AB method showed excellent applicability and it is simple, fast, non-toxic and suitable for high-throughput quantification, at least for biofilms grown in microtitre plates. It shows also great reproducibility and good sensitivity which is very important in antibiotic effect studies. To confirm the killing properties of the antibiotics used, the second method based on LIVE/DEAD biofilm staining was used. This use two stains: Syto9 (green fluorescence) and propionium iodide (PI – red fluorescence). Syto9 stains DNA in living cells while PI reduces green fluorescence only in dead cells. Fluorescence is observed with a confocal laser scanning microscope (CLSM).

Using those two methods authors showed that all SAMPs were clearly more effective in reducing metabolic activity in staphylococcal biofilms at low concentrations compared with antibiotics, even though they generally had higher MICs under planktonic growth conditions. In general, antibiotics were rarely able to cause a complete suppression of metabolic activity. In contrast, SAMPs were frequently able to suppress metabolic activity completely, indicating effective killing. It seems that SAMPs caused damage of the bacterial cell membranes even in slow growing or dormant bacteria embedded in a biofilm. In contrast, the antimicrobial agents used in this study predominantly affected growing bacteria by inhibiting their cell wall development (vancomycin) or by inhibition of their protein synthesis (linezolid, rifampicin and tetracycline).

In conclusion the SAMPs are potential new therapeutic agents in biofilm-associated infections. They could be especially attractive for topical treatment of chronic wound infections. The possible clinical applicability of SAMPs to prevent medical device-associated staphylococcal infections warrants future in vivo studies.

So maybe we can win this war…….

dr Jaroslaw E. Krol

UoI, Moscow

Functions of horizontally transferred genes

Horizontal gene transfer (HGT) is thought to play an important role in the evolution of species and innovation of genomes. Different researchers examined functional propensity in HGT of protein families.

First, Jain et al. (1999) proposed under the complexity hypothesis that HGT may have occurred preferentially among operational genes (those that maintain cell growth such as metabolism-related genes) than among informational genes (those involved in DNA replication, transcription, and translation) which are part of more complex protein-interaction networks.

Second, Nakamura et al. (2004) observed that only parts of genes in functional categories such as mobile element, cell surface, DNA binding, and pathogenicity-related, were preferred.

Third, Beiko et al. (2005) found extensive evidence for the preferential transfer of metabolic genes, while informational genes (e.g. ribosomal proteins, and proteins involved in DNA replication and repair, cell wall synthesis, and cell division) are susceptible or resistant to HGT.

Recently, Choi et al. (2007) suggested that there is no strong preference of HGT for protein families of particular cellular or molecular functions. They reconfirmed previous findings that HGT was biased toward cell surface and DNA binding functions (Nakamura et al., 2004), but the biases are marginal. They suggest that HGT is nearly neutral to all genes and that a random HGT process is followed by selection due to environment or other factors.

These discrepancies may be due to differences in the methods (e.g. phylogenetic versus compositional methods) and databases used, the genome samples tested, and possibly other reasons. For example, functions were assigned to protein families by using different databases: that is, (i) The Institute for Genomic Research role categories database (Peterson et al., 2001), (ii) The NCBI clusters of orthologous groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/), and (iii) Gene Ontology (GO) terms (Camon et al., 2003).

This has inspired us to examine functional correlates of the vertically versus horizontally transferred genes using uniform approaches (methods and databases).

REFERENCES:
Jain R, Rivera MC, Lake JA. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3801-6. Horizontal gene transfer among genomes: the complexity hypothesis.

Nakamura Y, Itoh T, Matsuda H, Gojobori T. Nat Genet. 2004 Jul;36(7):760-6. Biased biological functions of horizontally transferred genes in prokaryotic genomes.

Beiko RG, Harlow TJ, Ragan MA. Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14332-7. Highways of gene sharing in prokaryotes.

Choi IG, Kim SH. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4489-94. Global extent of horizontal gene transfer.

Dr. Haruo Suzuki
University of Idaho

Host roles in plasmid partitioning

In order to gain a clear idea of how plasmids or bacteria function on their own, it is necessary to have some understanding of how the two interact. There are, for example, obvious ways in which one influences the other, such as the conference of useful phenotypic traits (e.g. antibiotic resistance) to the host and the molecular maintenance (e.g. DNA replication) of plasmids by the host. There are also much more subtle interactions between the two, which are just now being elucidated for the first time. One such interaction is described by Kolatka et. al. in their recent publication.

This article describes the interactions of the broad-host-range IncP-1 plasmid RK2 and the partitioning systems of Pseudomonas putida and Escherichia coli. The authors found that the subcellular location of a given type of plasmid in a given strain of host will have a particular location within the cell. Also, and even more interestingly, this position depends on the protein products of both the host and plasmid.

Comparing the subcellular location of an RK2 mini-derivative in E. coli and P. putida showed this interaction between host and plasmid. Because the plasmid lacked an active partitioning system, the partitioning machinery of its host determined its position. In the case of E. coli, the plasmid was found to cluster at the cell poles, whereas in P. putida it was located either at a mid-cell or one quarter of the way into the cell on either side. The location within P. putida was explained by the interactions between the centromere-like sequences on the plasmid and the ParB protein encoded by the host’s chromosome. Indeed, when the parB gene was inserted into the E. coli chromosome the resultant partitioning mirrored that of P. putida. Conversely, when the par genes in P. putida where made nonfunctional then the plasmids were found at the poles. The position of RK2 itself showed similar locational disruptions when its position was determined in the P. putida par mutants. In all cases, plasmid location was determined by fluorescence in situ hybridization (FISH) and fluorescence microscopy. Protein interactions between the plasmids and bacteria were determined by formaldehyde cross-linking and chromatin immunoprecipitation.

It is shown here that, as always, the actions and activity of an individual organism (or even a mobile genetic element) are by no means completely independent, but rather that they are constantly influenced by the organisms and environments that they come into contact with. Indeed, the basic tenets of evolution involve, not isolated individuals, but the interaction between individuals. Therefore, in the case of bacteria and plasmids, it is necessary to not only study one and then the other, but also the influence that they have on one another.

Primary Article:

Kolatka, K., M. Witosinska, M. Pierechod, and I. Konieczny. 2008. Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2. Microbiology. 154:2847-56.

Additional References:

Ebersbach, G. & Gerdes, K. (2005). Plasmid segregation mechanisms. Annu Rev Genet 39, 453–479.

Funnell, B. E. (2005). Partition-mediated plasmid pairing. Plasmid 53, 119–125.

Gordon, S., Rech, J., Lane, D. & Wright, A. (2004). Kinetics of plasmid segregation in Escherichia coli. Mol Microbiol 51, 461–469.


Julie Hughes
Graduate Student
Department of Biological Sciences
University of Idaho

Piggery manure used for soil fertilization is a reservoir for
transferable antibiotic resistance plasmids

Chu Thi Thanh Binh, Holger Heuer, Martin Kaupenjohann & Kornelia Smalla
FEMS MICROBIOL. ECOL. 66:25-37

Overuse of antibiotics has been responsible for the spread of antibiotic resistance among bacteria all over the world. Continual use of antibiotics has maintained a strong selective pressure for the persistence of antibiotic resistance genes, while horizontal gene transfer has resulted in the spread of these genes across phylogenetically diverse bacteria (Witte, 1998; Rhodes et al., 2000; Schmidt et al., 2001; Tennstedt et al., 2003). Studies on plasmid content from manures have shown the presence of transferable plasmids carrying antibiotic resistance genes (Gotz et al., 1996; Smalla et al., 2000; Heuer et al., 2002; van Overbeek et al., 2002). This study looks at manures from 15 pig farms, where each farm represents a different size of herd or different quantity of meat production.

16 manure samples were taken from 15 different farms across Germany. Exogenous biparental matings were carried out in the laboratory by using E. coli CV601 as the recipient and manure as donor. Mixtures of recipient and donor were incubated overnight and then plated on agar supplemented with either amoxicillin, sulfadiazine or tetracycline.
A total of 228 transconjugants were picked. Eight antibiotics were tested on all transconjugants using the disc diffusion method (Barry et al.,). Based on different combinations of antibiotic resistance phenotypes, 37 unique patterns were found. 204 transconjugants showed sulfadiazine resistance. The frequent use of sulfadizine in animal husbandry may be the reason for this observation. 40 transconjugants showed resistance to six antibiotics and 4 were resistant to all 8 antibiotics used. This is a frightening scenario, since only 8 antibiotics were tested and many more resistance genes may be present on these plasmids. A previous study (Normark & Normark, 2002) had shown that selection for one antibiotic might co-select other antibiotics. The authors hypothesize that this may be the reason for the appearance of multiple antibiotic resistances on these plasmids. In order to make their study simpler, they decided to use a subset of the 228 transconjugants. Hence, one transconjugant was chosen per manure for each antibiotic resistance pattern. This gave them 81 plasmids which they decided to analyse further.
Plasmids extracted from transconjugants were dot-blotted and hybridized with probes specific for replicon sequences of the broad-host-range (BHR) plasmid classes IncN, IncW, IncP-1 and IncQ. 28 plasmids were found to be IncN, 1 was IncW, 13 were IncP-1, 19 were similar to the recently discovered pHHV216-like plasmids (Heuer et al., 2008) and 20 plasmids could not be assigned to any of the known Inc groups. Next the authors wanted to see which genes were conferring resistances to amoxicillin and sulfadiazine in these plasmids. Dot-blotted plasmid DNA was hybridized with labeled probes for bla-TEM, sul1, sul2 and sul3 genes. While bla-TEM genes are most often associated with resistance to amoxicillin, a combination of sul1, sul2 and sul3 genes may be responsible for sulfadiazine resistance. From this experiment they saw that all transconjugants with the amoxicillin resistance phenotype carried the bla-TEM gene, confirming the findings of Binh et al., who showed the frequent occurrence of bla-TEM genes in manure and amoxicillin resistance soils. An interesting observation was the repeated occurrence of these genes on similar plasmids, for example the occurrence of bla-TEM genes on all 28 IncN plasmids. The authors conclude that IncN plasmids that were captured from 10 different manures could be responsible for the dissemination of bla-TEM genes. Similarly, the sul2 gene was found on all 19 pHHV216-type plasmids captured from 6 manures and the sul1 gene was found on 12 of 13 IncP-1 plasmids. The authors state that their work shows that antibiotic resistance genes are associated preferably with BHR plasmids. Next the authors tested the transferability of the 81 plasmids by carrying out matings where they used their transconjugants as donors and E. coli J53 as the recipient. They found that 73 could be transferred to the recipient and only 8 could not. 6 of these 8 were the pHHV216-like plasmids.

In order to compare the method of direct PCR-based detection of plasmids in total DNA of manure to the method of plasmid capture, they used primers specific to repA for IncN, trfA2 for IncP-1, or oriV for IncQ and IncW for PCR of total DNA of manure.
No correlation was observed between the frequency of plasmid capture and plasmid abundance as noted from total DNA of manure. For example, although one third of the plasmids captured from 15 manures were characterized as IncN, this class of plasmid was detected in only 5 manures by PCR and Southern blot hybridization. The authors attribute this to the low abundance of IncN plasmids in manure, which could have resulted in making PCR based detection difficult. Using the exogenous plasmid isolation method, they were able to capture IncN plasmids from these soils. Thus, they suggest that the exogenous isolation method captures plasmids even when they are not abundant and PCR-based detection of plasmid types may not be as efficient.

This study is important because it shows how prevalent broad host range plasmids are. Moreover, association of antibiotic resistance genes with such plasmids ensures their rapid spread in an environment with antibiotics that maintain a strong selection. We get some idea of the prevalence of resistance to antibiotics in bacteria. All transconjugants were found to confer resistance to one or more antibiotics. Co-selection of antibiotics is also a phenomenon that we should be looking at closely.

References:

Witte W. (1998): Medical consequences of antibiotic use in agriculture. Science 279: 996–997.

Rhodes G., Huys G., Swings J., McGann P., Hiney M., Smith P. & Pickup R.W.
(2000): Distribution of oxytetracycline resistance plasmids between Aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890.

Schmidt A.S., Bruun M.S., Dalsgaard I. & Larsen J.L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67: 5675–5682.

Tennstedt T., Szczepanowski R., Braun S., Puhler A. & Schlüter A. (2003): Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252.

Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., van Elsas J.D. & Smalla K. (1996) Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62: 2621–2628.

Smalla K., Heuer H., Gotz A., Niemeyer D., Krogerrecklenfort E. & Tietze E., 2000: Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66: 4854–4862.

Heuer H., Krogerrecklenfort E., Egan S. et al. (2002): Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 28-302.

Van Overbeek L.S., Wellington E.M.H., Egan S., Smalla K., Heuer H., Collard J.M., Guillaume G., Karagouni A.D., Nikolakopoulou T.L. & van Elsas J.D. (2002): Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288.

Normark B.H. & Normark S (2002): Evolution and spread of antibiotic resistance. J Intern Med 252: 91–106.

Heuer H., Kopmann C., Binh C.T. T., Top E.M., Smalla K.(2008): Spreading antibiotic resistance through spread manure: characteristics 1 of a novel 2 plasmid type with low %G+C content. In press.

Barry A. L., Garcia F., and Thrupp L.D. (1970): An improved single-disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158.


Diya Sen
Graduate Student,
Department of Biological Sciences,
University of Idaho

Lessons from the first comprehensive survey of prokaryote genomics

Genomics of bacteria and archaeal: the emerging dynamic view of the prokaryotic world. Eugene V. Koonin & Yuri I. Wolf, Nucleic Acids Research (2008)

The first bacterial genome (Haemophilus influenzae) was published in 1995, ushering in the so-called age of genomics. Since then, exponentially increasing numbers of whole-genome sequencing projects have generated a huge amount of raw data. While this holds great promise for developing our understanding of how prokaryotic genomes are formed and function, extracting meaningful observations from that mountain of data is a big challenge.

Drs Eugene Koonin and Yuri Wolf recently tackled this daunting task and embarked on a comprehensive survey of the genomic data produced to date by microbial sequencing projects. Their latest paper, titled “Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world”, presents their findings in a dense but rich monograph that offers deep insight into processes of genome evolution and defines general principles of prokaryotic genome organization.

Although there can be no substitute for reading the original paper, a point-by-point summary of the paper that may interest readers is provided here.

Dr. Geraldine A. Van der Auwera
Harvard Medical School

Bacteriophages SPP1 contains two tail tube proteins produced by programmed translational frameshift

Origin and function of the two major tail proteins of bacteriophage SPP1. Auzat et al., Molecular Microbiology (2008) 70, 557-569


Bacteriophages are viruses that infect bacteria and are known to play roles in horizontal gene transfer. The majority of known bacteriophages have a head and long non-contractile tail that serves as a pipeline for phage genome delivery into bacterial cell. Here, the authors report that the tail tube of Bacillus subtilus bacteriaphage SPP1 is comprised of two proteins, gp17.1 and gp17.1* , that are produced by a translational frameshift. This mosaic construction of tail tube was found to be important for assembly of the functional tail tube, but its significance is not fully uncovered yet.

Previously, it was shown that the key event of phage DNA injection in bacterial cell is a rearrangement of the inner wall of the tail tube (EMBO J, (2007) 26, 3720-3728). In this article, the authors separated SSP1 tail proteins by SDS-PAGE and found a protein band which was not expected from phage DNA sequence. Protein sequencing analysis indicated that the unexpected protein designated gp17.1* has the same amino-terminal sequence as that of tail protein gp17.1. gp17.1* is 10 kDa larger than gp17.1. The tail tube is made up of these two proteins at the ratio of 1:3. Based on the molecular size of gp17.1* and the sequences of gene 17.1, authors postulated that 17.1* is produced by a translational frameshift. Using site-directed mutagenesis of coding sequence 17.1 with protein profile analysis of the mutant phages, authors found out that 5'-CCCUAA-3' sequence located at the end of coding sequence 17.1 was the frameshift position.

To get insight into the function of gp17.1*, the authors constructed mutant phages that have tail tubes comprised exclusively of gp17.1 or gp17.1* and analyzed their structures by electron microscopy. When phages are assembled under the condition which either gp17.1 or gp17.1* are exclusively expressed, significant numbers of tailless heads (capsids) are made. This suggests the 3:1 ratio of gp17.1 and gp17.1* is important for correct phage assembly. Interestingly, both mutant phages had infection activity. The length and flexibility of mutant tails composed of either gp17.1 or gp17.1* were identical to SSP1 wild-type tails. gp17.1*-specific tail has protrusions on surface while gp17.1-specific tail has smooth surface. Authors postulate that carboxyl-terminus of gp17.1* causes protrusions on tail surface which facilitate initial contact of phages and attachment to the bacterial surface, while gp17.1 is ensures correct assembly of tail tube.

The potential translational frameshift site producing a carboxyl-terminus extension in a protein are also found in other phage surface protein genes (Mol Microbiol. (2003) 50, 303-317). Why do phages need two types of surface proteins which are identical except the carboxyl end extension of one of the proteins? What is the significance of strict ratio of the two proteins? Why is this the best strategy for phages? What is the target of the protrusion on tail tube surface? These are still interesting mysteries. It might be interesting to compare the host ranges of mutant phages that have either one of the two surface proteins.


References
Origin and Function of the Two Major Tail Proteins of Bacteriophage SPP1. Auzat I, Dröge A, Weise F, Lurz R, Tavares P. Molecular Microbiology (2008) 70: 557-569

Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection. Plisson C, White HE, Auzat I, Zafarani A, São-José C, Lhuillier S, Tavares P, Orlova EV,
EMBO J (2007) 26:3720-8.

Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed + 1 translational frameshifting in structural protein synthesis. Zimmer M, Sattelberger E, Inman RB, Calendar R, Loessner MJ., Molecular Microbiology. (2003) 50:303-317.


Hirokazu Yano (university of idaho)