In the process of CRISPR adaptation, short pieces of DNA ("spacers") are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of "self" is mediated by the RecBCD dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers from both high copy plasmids and phages.

Multidrug resistance (MDR) translocators recently identified in bacteria constitute an excellent model system for studying the MDR phenomenon and its clinical relevance. Here we describe the identification and characterization of an unusual MDR gene (mdfA) from Escherichia coli. mdfA encodes a putative membrane protein (MdfA) of 410 amino acid residues which belongs to the major facilitator superfamily of transport proteins. Cells expressing MdfA from a multicopy plasmid are substantially more resistant to a diverse group of cationic or zwitterionic lipophilic compounds such as ethidium bromide, tetraphenylphosphonium, rhodamine, daunomycin, benzalkonium, rifampin, tetracycline, and puromycin. Surprisingly, however, MdfA also confers resistance to chemically unrelated, clinically important antibiotics such as chloramphenicol, erythromycin, and certain aminoglycosides and fluoroquinolones. Transport experiments with an E. coli strain lacking F 1 -F 0 proton ATPase activity indicate that MdfA is a multidrug transporter that is driven by the proton electrochemical gradient.The simultaneous emergence of resistance of eukaryotic and prokaryotic cells to many unrelated hydrophobic chemotherapeutic drugs is termed multidrug resistance (MDR). Recently, it has become evident that a variety of MDR efflux systems are widely distributed among prokaryotic microorganisms, including pathogenic bacteria (18,29,30). They belong to three different families of transport proteins: the major facilitator superfamily (MFS) (22), the resistance nodulation division (RND) family (28, 32), and the small multidrug resistance (SMR) family of small translocases (31). Broad substrate specificity and the ability of the prokaryotic MDR transporters to extrude a variety of unrelated drugs from the cell are similar to P-glycoprotein-mediated MDR in mammalian systems (9, 10). Importantly, however, unlike mammalian P glycoprotein, which belongs to the ATP binding cassette (ABC) superfamily of ATP-dependent transport proteins (15), the known bacterial MDR transporters probably utilize the membrane potential (⌬) and/or the proton gradient (⌬pH) as the driving force for drug export (6,12,38). There is also experimental evidence for putative ATP-dependent MDR systems in bacteria (5), but these proteins have not been characterized. Overall, it is likely that in the future, strategies for antibacterial chemotherapy will confront an increasing number of nonspecific mechanisms of resistance (27).In addition to their potential clinical importance, the nonspecific MDR proteins pose intriguing and challenging questions concerning the mechanism of action of transport systems. While the substrate specificity of the mammalian P glycoprotein is probably limited to lipophilic compounds, most of which are protonated at physiological pH, there are bacterial MDR proteins that recognize antibiotics which are uncharged or zwitterionic at a neutral pH, in addition to the lipophilic drugs (25,26).Many of the bacterial drug translocators belong to the MFS of transport pro...

With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an Ϸ100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.bacteriophage T7 ͉ BirA ͉ Escherichia coli ͉ water sample T he number and diversity of bacteriophages in the environment provide a promising natural pool of specific detection tools for pathogenic bacteria. Currently there are several phagebased methods for detection of pathogenic bacteria (1): a plaque assay for detection of Mycobacterium tuberculosis (2); fluorescence-labeled phage and immunomagnetic separation assay for detection of Escherichia coli O157:H7 (3, 4); phage-based electrochemical assays (5); a luciferase reporter mycobacteriophage and Listeria phage assays (6, 7); and detection of the phagemediated bacterial lysis and release of host enzymes (e.g., adenylate kinase) (8).Two limiting features when detecting pathogenic bacteria are sensitivity and rapidity. Common fluorophores (e.g., GFP and luciferase) used as reporters have two major disadvantages: low signal-to-noise ratio due to autofluorescence of clinical samples and of bacterial cells and low photostability, such as fast photobleaching. To overcome these disadvantages, we used new fluorescent semiconductor nanocrystals, quantum dots (QDs) (9). QDs are colloidal semiconductor (e.g., CdSe) crystals of a few nanometers in diameter. They exhibit broadband absorption spectra, and their emissions are of narrow bandwidth with size-dependent local maxima. The presence of an outer shell of a few atomic layers (e.g., ZnS) increases the quantum yield and further enhances the photostability, resulting in photostable fluorescent probes superior to conventional organic dyes. Recently, development in surface chemistry protocols allows conjugation of biomolecules onto these QDs to target specific biological molecules and probe nanoenvironments (10-12). The power to observe and trace single QDs or a group of bioconjugated QDs, enabling more precise quantitative biology, has been claimed to be one of the most exciting new capabilities offered to biologists today (13,14).Typically, the detection of small numbers of bacteria in environmental or clinical samples require...

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.

We show that phage lysogenization, lysogens, and prophage induction are all targeted by CRISPR. The results demonstrate that genomic DNA is not immune to the CRISPR system, that the CRISPR system does not require noncytoplasmic elements, and that the system protects from phages entering and exiting the lysogenic cycle.The CRISPR system was recently identified as an adaptive defense mechanism against bacteriophages and extrachromosomal elements. The function of the CRISPR system in antiviral defense was demonstrated experimentally for the first time in 2007 by Barrangou et al. for Streptococcus thermophilus (3). CRISPR protection from lytic proliferation of phages and horizontal gene transfer has since been reported for different bacterial species (5,11,17). However, studies of the CRISPR response to temperate phages are scarce. It has been shown that Streptococcus pyogenes strains harboring a CRISPR system contain few or no prophages, whereas strains lacking a CRISPR system are polylysogens. It has also been shown that strains harboring spacers against prophages are free of these prophages (4,7,9,12). Another study has shown that the presence of two spacers against a phage of Pseudomonas aeruginosa, DMS3, inhibits biofilm formation and swarming motility of the lysogen in a CRISPR-dependent manner (20). This phenomenon shown for P. aeruginosa demonstrates a function of the CRISPR system in controlling group behavior responses of a lysogen by an uncharacterized mechanism. Nevertheless, experimental evidence of CRISPR protection from lysogenization and lysogens, which may explain the mutually exclusive relationship between CRISPR spacers and prophages, has not yet been demonstrated.The CRISPR activity demonstrated thus far against plasmid transfer or lytic growth of phages involves passage of the alien DNA through membranes. It is still unknown whether this membrane passage is essential for the activity of the CRISPR system, which perhaps requires involvement of a membrane or periplasmic protein(s). A mechanism requiring such proteins should be excluded if the CRISPR system were shown to act on prophages already present in the cytoplasm. Showing CRISPR activity against an integrated prophage will also address whether the bacterial genome is protected from the CRISPR system. Recent findings show that small parts of the CRISPR system-CRISPR arrays-are protected by virtue of their flanking repeats, at least in Staphylococcus epidermidis (13). Activity of the CRISPR system against a prophage integrated in the genome would suggest that the genomic DNA is not recognized as "self" due to unique modifications, as is the case with protection from restriction endonucleases (14). In this short report, several remarkable aspects of CRISPR-mediated protection are revealed. (i) Lysogenization is prevented by the CRISPR system. (ii) Integrated prophage is targeted by CRISPR, resulting in cellular death. (iii) The CRISPR system can rescue bacteria from prophage induction. The results also demonstrate that the CRISPR system is acti...

Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

SummaryThe poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage l DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of l DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to l's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.

Bacterial nucleoid organization is believed to have minimal influence on the global transcription program. Using an altered bacterial histone-like protein, HU␣, we show that reorganization of the nucleoid configuration can dynamically modulate the cellular transcription pattern. The mutant protein transformed the loosely packed nucleoid into a densely condensed structure. The nucleoid compaction, coupled with increased global DNA supercoiling, generated radical changes in the morphology, physiology, and metabolism of wild-type K-12 Escherichia coli. Many constitutive housekeeping genes involved in nutrient utilization were repressed, whereas many quiescent genes associated with virulence were activated in the mutant. We propose that, as in eukaryotes, the nucleoid architecture dictates the global transcription profile and, consequently, the behavior pattern in bacteria.bacterial HU ͉ nucleoid condensation ͉ virulence T he highly organized eukaryotic chromosome requires elaborate remodeling for coordination of transcription processes. The bacterial chromosome, however, constitutes a comparatively open and expanded structure, accessible throughout the cell cycle to DNA-binding proteins, polymerases, and ribosomes (1, 2). The apparent lack of a systemic hierarchy of nucleoid organization in bacteria was attributed to a low stability of histone-like protein-DNA complexes and the dynamic nature of the bacterial chromosome (3). In the absence of any perceptible higher-order chromosome organization imposing a general level of restriction on the accessibility of bacterial promoters, gene expression is believed to be regulated by operon-specific factors, adjacent DNA control elements and local DNA architecture; global nuclear organization is thought to contribute minimally to overall control of cellular processes involving DNA as substrate (4). Considering the exquisite precision with which bacteria can modulate their gene-expression profile to various environmental challenges, the ostensible lack of influence of chromosome organization over global gene regulation is surprising.Based on its small size, basic nature, cellular abundance, and sequence-independent DNA-binding capacity, the nucleoidassociated protein HU has long been characterized as the bacterial counterpart of eukaryotic histones (5). HU was initially attributed with the capability to form nucleosome-like structures in bacterial chromosomes (6), but subsequent studies have resulted in conflicting reports about the exact role of HU in chromosome compaction (7,8). In almost all bacteria except enterobacteriaciae, including Eschericiha coli, HU exists as an 18-kDa homodimer. In E. coli, HU is a heterodimer of two subunits, HU␣ and HU␤.Using a gain-of-function HU␣ mutant, we demonstrate that nucleoid structural reorganization in bacteria can directly induce a radical change in the gene-expression profile, resulting in dramatic changes in cellular morphology and physiology. Materials and MethodsBacterial Strains, Media, and Growth Conditions. Mutagenesis of HU␣ ...