Reduction of the nosocomial meticillin-resistant Staphylococcus aureus incidence density by a region-wide search and follow-strategy in forty German hospitals of the EUREGIO, 2009 to 2011.

Meticillin-resistant Staphylococcus aureus (MRSA) disseminates between hospitals serving one patient catchment area. Successful prevention and control requires concerted efforts and regional surveillance. Forty hospitals located in the German EUREGIO have established a network for combating MRSA. In 2007 they agreed upon a synchronised strategy for screening of risk patients and a standard for transmissionbased precautions (search and follow). The same year, the hospitals started synchronised MRSA prevention and annually reporting MRSA-data to the public health authorities. The median rate of screening cultures per 100 patients admitted increased from 4.38 in 2007 to 34.4 in 2011 (p<0.0001). Between 2007 and 2011, the overall incidence density of MRSA (0.87 MRSA cases/1,000 patient days vs 1.54; p<0.0001) increased significantly. In contrast, both the incidence density of nosocomial MRSA cases (0.13 nosocomial MRSA cases/1,000 patient days in 2009 vs 0.08 in 2011; p=0.0084) and the MRSA-days-associated nosocomial MRSA rate (5.51 nosocomial MRSA cases/1,000 MRSA days in 2009 vs 3.80 in 2011; p=0.0437) decreased significantly after the second year of the project. We documented adherence to the regional screening strategy resulting in improved detection of MRSA carriers at admission. Subsequently, after two years the nosocomial MRSA-incidence density was reduced. Regional surveillance data, annually provided as benchmarking to the regional hospitals and public health authorities, indicated successful prevention.

Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses.

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V.harveyi ATTC BAA-1116 and V.campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T=12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.

Jumbo bacteriophages.

There is currently a handful of genome sequences available for tailed bacteriophages with genomes of more than 200 kbp of DNA, designated here as giant or jumbo phages. The majority of the proteins predicted from the genome sequences of these phages have no matches in the current sequence databases, and the genomes themselves are diverse enough to preclude the sorts of detailed comparative analysis that has benefited study of the smaller phages, for which hundreds of genome sequences are available. However, it is informative to extrapolate the better known genome organizations and mechanisms of evolution seen in the smaller phages to the jumbo phages. In this way, we see that the jumbo phages encode the same functions as the smaller phages, supplemented with large numbers of mostly small genes of mostly undiscovered functions. A case can be made that the jumbo phages evolved from smaller tailed phages, possibly in a process mediated by the constraints imposed on genome size by capsid size.

Bacteriophage lambda PaPa: not the mother of all lambda phages.

The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

Topologically linked protein rings in the bacteriophage HK97 capsid.

The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

Virus maturation involving large subunit rotations and local refolding.

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

Evolution: the long evolutionary reach of viruses.

The structure of a phage capsid protein provides good evidence this phage shares ancestry with an animal virus. In this and similar cases, either the viral lineages predate the emergence of the three contemporary domains of life, or viruses have been leaping the phylogenetic chasms that separate the domains.

Bacteriophage HK97 head assembly.

The head assembly pathway of bacteriophage HK97 shares many features with head assembly pathways determined for other dsDNA phages, and it also provides examples of novel variations on the basic theme. We describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. Comparisons of head assembly pathways among different phages, as well as comparisons of the organization of the genes that specify those pathways, suggest the range of different solutions phages have found to common assembly problems and give insight into the evolutionary histories of these assembly processes.

Where are the pseudogenes in bacterial genomes?

Most bacterial genomes have very few pseudogenes; notable exceptions include the genomes of the intracellular parasites Rickettsia prowazekii and Mycobacterium leprae. As DNA can be introduced into microbial genomes in many ways, the compact nature of these genomes suggests that the rate of DNA influx is balanced by the rate of DNA deletion. We propose that the influx of dangerous genetic elements such as transposons and bacteriophages selects for the maintenance of relatively high deletion rates in most bacteria; the sheltered lifestyle of intracellular parasites removes this threat, leading to reduced deletion rates and larger pseudogene loads.

The origins and ongoing evolution of viruses.

Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.

Assembly in vitro of bacteriophage HK97 proheads.

Bacteriophage HK97 is a lambdoid phage with a head assembled from 415 copies of a 42 kDa subunit arranged in an icosahedrally symmetrical lattice with a triangulation number of 7. Prohead I, the first shell structure in the assembly pathway, is composed of 42 kDa coat protein subunits that have not yet undergone the proteolytic cleavage, conformational changes, and covalent cross-linking steps that occur later in the assembly of mature heads. Prohead I can be efficiently dissociated into capsomeres by treatment with 2 M KCl. The resulting capsomeres are a mixture of two species, identified as pentamers and hexamers of the 42 kDa subunit. These capsomeres were also detected as the products of chaperonin-assisted renaturation of 42 kDa polypeptide in vitro at room temperature or in the course of self folding and assembly in vitro at 0 degrees C. Pentamer and hexamer capsomeres can be interconverted in vitro by manipulating solvent conditions, and this makes it possible to carry out the in vitro shell assembly reaction at different input ratios of hexamer to pentamer. The Prohead I structures produced are always the normal (T = 7) size regardless of the input pentamer to hexamer ratio. Assembly is most efficient when the pentamer to hexamer ratio is 1:5 (a mass ratio of 1:6), or the same as the capsomere ratio in a T = 7 shell.

Genetic basis of bacteriophage HK97 prohead assembly.

We report studies to determine which bacteriophage genes are required for assembly of phage HK97 proheads and what roles they play. We identify the gene encoding the major capsid protein of phage HK97 and report its DNA sequence, together with the DNA sequences of the two genes immediately upstream from it. When the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of the unprocessed 42 kDa capsid protein. No separately encoded scaffolding protein is required for this assembly. If the 25 kDa product of the next gene upstream is co-expressed with the capsid protein, the prohead structures that are produced undergo the normal morphogenetic cleavage, which removes 102 amino acids from the N terminus of each subunit, leaving 31 kDa subunits. The 25 kDa protein is therefore probably a phage-encoded protease. The third gene, upstream from the protease gene, encodes the portal protein. Presence of the portal protein is not required for assembly of the capsid protein. Analysis of the phenotypes of four single amino acid-substitution mutants in the capsid-protein gene leads to several insights into the functions of the capsid protein and its interactions with the putative protease.

A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein.

Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames. The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate. Our results together with those of Katsura and Kühl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.

Domain structure and quaternary organization of the bacteriophage P22 Erf protein.

The structure and activities of the recombination-promoting P22 Erf protein were examined in vitro. Treatment of the protein with elastase produces a stable amino-terminal fragment, consisting of amino acid residues 1 to (approximately) 136. We have purified this fragment, designated fragment B, to apparent homogeneity by gel filtration chromatography. Fragment B retains the oligomeric structure and single-stranded DNA binding specificity of intact Erf. It differs, however, in lacking the ability of intact Erf to bind single-stranded DNA into large aggregates following mild heat treatment of the protein. In addition, its binding to DNA may be weaker than that of intact Erf. Intact Erf sediments through a sucrose gradient as a discrete species with an apparent S20,w of approximately 11 X 7 S. Its sedimentation behavior is affected little, if at all, by concentration. Fragment B also sediments as a discrete species at approximately 10 X 4 S. In the electron microscope, intact Erf appears as rings, with 10 to 14 small projecting structures resembling the teeth of a gear. Fragment B is similar, except that it appears to lack the peripheral structures. From these observations, we conclude that Erf consists of at least two structurally and functionally distinct domains, and that it has a discrete ring-like oligomeric structure.

Structural transitions during bacteriophage HK97 head assembly.

Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein.

Four amber fragments of the recombination-promoting P22 Erf protein were characterized. The intact Erf monomer contains 204 amino acids. The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in vivo. The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf. It breaks down to a stable remnant that is slightly larger than the 149 residue fragment. The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf. Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0. The 95 residue fragment is unstable in vivo. These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNA-binding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo.

Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages.

We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), double-stranded DNA bacteriophages of Escherichia coli and members of the lambdoid or lambda-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or, in some cases, at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome. The comparative analysis also allows some inferences about function of genes or other genetic elements. We give examples for the generalized recombination genes of HK97, HK022 and P22, and for a putative headtail adaptor protein of HK97 and HK022. We also use the comparative approach to identify a new class of genetic elements, the morons, which consist of a protein-coding region flanked by a putative delta 70 promoter and a putative factor-independent transcription terminator, all located between two genes that may be adjacent in a different phage. We argue that morons are autonomous genetic modules that are expressed from the repressed prophage. Sequence composition of the morons implies that they have entered the phages' genomes by horizontal transfer in relatively recent evolutionary time.

Genomic sequence and analysis of the atypical temperate bacteriophage N15.

N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.