A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.

Characterization of Nda2, a plastoquinone-reducing type II NAD(P)H dehydrogenase in chlamydomonas chloroplasts.

Electron transfer pathways associated to oxygenic photosynthesis, including cyclic electron flow around photosystem I and chlororespiration, rely on non-photochemical reduction of plastoquinones (PQs). In higher plant chloroplasts, a bacterial-like NDH complex homologous to complex I is involved in PQ reduction, but such a complex is absent from Chlamydomonas plastids where a type II NAD(P)H dehydrogenase activity has been proposed to operate. With the aim to elucidate the nature of the enzyme-supporting non-photochemical reduction of PQs, one of the type II NAD(P)H dehydrogenases identified in the Chlamydomonas reinhardtii genome (Nda2) was produced as a recombinant protein in Escherichia coli and further characterized. As many type II NAD(P)H dehydrogenases, Nda2 uses NADH as a preferential substrate, but in contrast to the eukaryotic enzymes described so far, contains non-covalently bound FMN as a cofactor. When expressed at a low level, Nda2 complements growth of an E. coli lacking both NDH-1 and NDH-2, but is toxic at high expression levels. Using an antibody raised against the recombinant protein and based on its mass spectrometric identification, we show that Nda2 is localized in thylakoid membranes. Chlorophyll fluorescence measurements performed on thylakoid membranes show that Nda2 is able to interact with thylakoid membranes of C. reinhardtii by reducing PQs from exogenous NADH or NADPH. We discuss the possible involvement of Nda2 in cyclic electron flow around PSI, chlororespiration, and hydrogen production.

Agrobacterium tumefaciens type II NADH dehydrogenase. Characterization and interactions with bacterial and thylakoid membranes.

Type II NADH dehydrogenases (NDH-2) are monomeric enzymes that catalyse quinone reduction and allow electrons to enter the respiratory chain in different organisms including higher plant mitochondria, bacteria and yeasts. In this study, an Agrobacterium tumefaciens gene encoding a putative alternative NADH dehydrogenase (AtuNDH-2) was isolated and expressed in Escherichia coli as a (His)6-tagged protein. The purified 46 kDa protein contains FAD as a prosthetic group and oxidizes both NADH and NADPH with similar Vmax values, but with a much higher affinity for NADH than for NADPH. AtuNDH-2 complements the growth (on a minimal medium) of an E. coli mutant strain deficient in both NDH-1 and NDH-2, and is shown to supply electrons to the respiratory chain when incubated with bacterial membranes prepared from this mutant. By measuring photosystem II chlorophyll fluorescence on thylakoid membranes prepared from the green alga Chlamydomonas reinhardtii, we show that AtuNDH-2 is able to stimulate NADH-dependent reduction of the plastoquinone pool. We discuss the possibility of using heterologous expression of NDH-2 enzymes to improve nonphotochemical reduction of plastoquinones and H2 production in C. reinhardtii.

Plasticity of the gene functions for DNA replication in the T4-like phages.

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.

The diversity and evolution of the T4-type bacteriophages.

Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses.

Snapshot of the genome of the pseudo-T-even bacteriophage RB49.

RB49 is a virulent bacteriophage that infects Escherichia coli. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the approximately 170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.