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Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

Objectives: Treatment of infections caused by Acinetobacter baumannii nosocomial strains has become increasingly problematic owing to their resistance to antibiotics. ppGpp is a secondary messenger involved in growth control and various stress responses in bacteria. The mechanism for inhibition of antibiotic resistance via ppGpp is still unidentified in various pathogenic bacteria including A. baumannii. Here, we investigated the effects of ppGpp on efflux pump (EP)-related genes in A. baumannii.Methods: ppGpp-deficient and -complementary strains were constructed by conjugation and we confirmed (p)ppGpp measurements by thin-layer chromatography. We observed that the ppGpp-deficient strain (ΔA1S_0579) showed abnormal stretching patterns by transmission electron microscopy analysis. The MICs of antimicrobial agents for the WT A. baumannii (ATCC 17978), ppGpp-deficient and complementary strains were determined by the Etest and broth dilution assay methods. The expression levels of EP-related genes were determined by quantitative RT-PCR.Results: We observed morphological differences between a ppGpp-deficient strain (ΔA1S_0579) and the WT strain. Dramatic reductions of MICs in the ppGpp-deficient strain compared with the WT were observed for gentamicin (2.6-fold), tetracycline (3.9-fold), erythromycin (4-fold) and trimethoprim (>4-fold). Expression of the EP-related genes abeB (2.8-fold), tet(A) (2.3-fold), adeB (10.0-fold), adeI (9.9-fold), adeJ (11.8-fold) and adeK (14.4-fold) was also decreased in the ppGpp-deficient strain.Conclusions: This study demonstrates that ppGpp regulates EP-related gene expression in A. baumannii, affecting antibiotic susceptibility. To date, treatment for MDR A. baumannii has had no new antimicrobial agents, so the A1S_0579 gene could be a novel therapeutic target for rational drug design by affecting ppGpp production. [ABSTRACT FROM AUTHOR]

Summary Transcription start points in bacteria are influenced by the nature of the RNA polymerase·promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing σ70, it is presumed that specific sequence in one or more of the −10, extended −10 and −35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF. [ABSTRACT FROM AUTHOR]