A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes.

Shiga toxin-producing Escherichia coli (STEC) colonizes the human intestine, causing haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Treatment options are limited to intravenous fluids in part because sublethal doses of some antibiotics have been shown to stimulate increased toxin release and enhance the risk of progression to HUS. Preventative antimicrobial agents, especially those that build on the natural antimicrobial action of the host defence, may provide a better option. In order to survive the acid stress of gastric passage, STEC is equipped with numerous acid resistance and DNA repair mechanisms. Inhibition of acid-induced DNA repair may offer a strategy to target survival of ingested STEC. We report here that brief pretreatment with a novel antimicrobial peptide, which was previously shown to inhibit bacterial DNA repair, significantly and profoundly reduces survival of acid-stressed O157 : H7 and non-O157 : H7 STEC seropathotypes that are highly associated with HUS. Reduction in survival rates of STEC range from 3 to 5 log. We also show that peptide/acid treatment results in little or no increase in toxin production, thereby reducing the risk of progression to HUS. This study identifies the peptide wrwycr as a potential new candidate for a preventative antimicrobial for STEC infection.

Production of shotgun libraries using random amplification.

In the following report, thermal cycling coupled with random 10-mers as primers was used to construct randomly amplified shotgun libraries (RASLs). This approach allowed shotgun libraries to be constructed from nanogram quantities of input DNA. RASLs contained inserts from throughout a target genome in an unbiased fashion and did not appear to contain chimeric sequences. This protocol should be useful for shotgun sequencing the genomes of unculturable organisms and rapidly producing shotgun libraries from cosmids, fosmids, yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs).

Characterization of a mutation of bacteriophage lambda integrase. Putative role in core binding and strand exchange for a conserved residue.

Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics. The bacteriophage lambda-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways. We have characterized a mutant version of lambda Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways. Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination. Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes. In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions. Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation. Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.

Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.

The amino terminus of bacteriophage lambda integrase is involved in protein-protein interactions during recombination.

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.

In vitro selection of integration host factor binding sites.

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.

Analysis of higher order intermediates and synapsis in the bent-L pathway of bacteriophage lambda site-specific recombination.

The integrase protein of bacteriophage lambda mediates recombination via four distinct pathways. The recent in vitro reconstitution of the efficient bidirectional reaction between two attLtenP'1 target sites now allows comparisons of this pathway, known as the bent-L pathway, with the inefficient bidirectional straight-L pathway and with the efficient but unidirectional pathways of integration and excision. To this end, a series of higher order intermediates of the bent-L pathway was characterized using gel mobility shift assays, two-dimensional gel analysis, and footprinting. The analysis spans the initial binding of proteins to individual DNA target sites, synapsis of two partner DNA targets, and strand exchange. This study identifies a presynaptic "checkpoint" of recombination. It shows that synapsis is a slow step in the recombination reaction, while subsequent strand exchange is comparatively fast. Synaptic complexes contain a preponderance of recombinant products, suggesting that an energetically favorable but somewhat subtle conformational change drives strand exchange. In addition, comparison of wild-type integrase with a catalytically defective mutant of integrase, IntF, showed that, in addition to the catalysis defect, this mutant has different DNA-binding properties than the wild-type protein.

Architectural flexibility in lambda site-specific recombination: three alternate conformations channel the attL site into three distinct pathways.

BACKGROUND: In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo, recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5-10-fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro. RESULTS: We find that IHF can stimulate the in vitro recombination between two attLs that are modified to be defective in one of the high affinity binding sites for Int, P'1. With such substrates, the efficiency of IHF-stimulated recombination is comparable to that seen in vivo. The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo, but that some unknown factor or condition permits it to operate on wild-type as well as mutated attL sites. CONCLUSIONS: The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P'1 site. In addition to demonstrating the architectural flexibility of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.

Exclusive breast-feeding duration is associated with attitudinal, socioeconomic and biocultural determinants in three Latin American countries.

International health organizations have recommended exclusive breast-feeding (EBF) (i.e., breast milk as the only source of food) as the optimal infant feeding method during the first 4-6 mo of life. Therefore, it is important to document the determinants of EBF in different populations. Low-income urban women from Brazil (n = 446, 2 maternity wards), Honduras (n = 1582, 3 maternity wards) and Mexico (n = 765, 3 maternity wards) were interviewed at birth and in their homes at 1 mo and 2-4 mo after delivery. Multivariate survival analyses (Cox model) indicated that planned duration of EBF (all 3 countries), having a female infant, and not being employed (Brazil and Honduras), lower socioeconomic status (Honduras and Mexico) and higher birth weight (control hospital in Brazil and Honduras) were positively associated (P < or = 0.10) with EBF. Women who delivered in the maternity wards that had more developed breast-feeding promotion programs were more successful with EBF. The association between maternal education and EBF was modified by the maternity ward in Mexico and Honduras. Being > or = 18 y and having a partner living (Brazil) or not (Mexico) living at home were positively associated with EBF. These findings can contribute toward the design of EBF promotion efforts in Latin America.

Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins.

Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination. HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity. We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase. The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA. We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure. We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome. HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions. These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts. Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA. While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies. This probably reflects subtle structural differences between the assembled complexes.

Approaches to half-tetrad analysis in bacteria: recombination between repeated, inverse-order chromosomal sequences.

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.

Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation.

Sequences placed in inverse order at particular chromosome sites (permissive) recombine to generate an inversion; the same sequences, placed at other sites (nonpermissive) interact recombinationally but do not form the expected inversion recombinants. We have investigated the events that occur between sequences at nonpermissive sites. Genetically marked lac operons in inverse order were placed at nonpermissive sites in a single chromosome and Lac+ recombinants were selected. No inversions were formed. The Lac+ recombinants recovered include double-recombinant types in which information appears to have undergone a nonreciprocal information exchange; one mutant copy is repaired with no alteration of the other copy. Recombination within the lac operon is stimulated more than 100-fold by the presence of extensive homology (antenna sequences) outside of the region for which recombination is selected. Sequences placed in direct order at the ends of the same noninvertible chromosome segment recombine to form all the expected recombinant types including those in which a reciprocal exchange has generated a duplication. All the detected recombinant types can be accounted for by recombination between sister chromosomes. These results are discussed in terms of two alternative models. One explanation of the failure to detect inversion of some intervals is that particular inversions are lethal, despite the fact that no essential sequences are disrupted. Another explanation is that chromosome topology prevents sequences at nonpermissive sites in a single chromosome from engaging in the direct interaction required for inversion formation, but allows the sister strand exchanges that can generate the recombinant observed.

Characterization of a Marine Bacterium Associated with Crassostrea virginica (the Eastern Oyster).

A gram-negative bacterium found to be closely associated with oysters has been isolated and characterized. The organism, designated LST, has a generation time of 106 min in Marine broth under optimal growth conditions at 25 degrees C. During the decline phase of growth, it exhibits a morphological transition from a motile rod (ca. 1 mum in length) to an elongated, 3- to 40-mum, nonmotile, tightly coiled helix. LST synthesizes and releases a pigment in the stationary and decline phases of growth. Identified as melanin on the basis of chemical properties and UV absorbance maxima, the pigment comprises polymers of heterogeneous molecular weights, ranging from 12,000 to 120,000. The guanosine-plus-cytosine content of the LST DNA is 46%, and results of phenetic analysis and DNA-DNA hybridization indicate that this bacterium represents a new species. LST adheres to a variety of surfaces, including glass, plastics, and oyster shell, and has been shown to promote the settlement of oyster larvae.