Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family.

Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.

Diversity and antimicrobial resistance of Salmonella enterica isolates from surface water in Southeastern United States.

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA's Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.

Antibodies specific for nucleic acids and applications in genomic detection and clinical diagnostics.

Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies.

Detection of live Escherichia coli O157:H7 cells by PMA-qPCR.

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6.

The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C.

BACKGROUND: The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. RESULTS: Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. CONCLUSIONS: Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway.

An antibody-based microarray assay for small RNA detection.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction.

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.

A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum.

MinD is a key component of an oscillatory system that spatially regulates cell division in Escherichia coli. It is a peripheral membrane ATPase that recruits MinC and oscillates between the two halves of the cell in a MinE dependent manner. In vitro MinD binds to phospholipid vesicles in an ATP-dependent manner and is released through MinE-stimulated ATP hydrolysis. In this study we examined the function of the conserved C-terminus of MinD. Short truncations of three and ten amino acids dramatically decreased the ability of MinD to localize to the membrane and spatially regulate division. These truncations bound MinC but were deficient in targeting MinC to the septum. In vitro they dimerized, but were deficient in binding to phospholipid vesicles and undergoing MinE stimulation. We suggest a model in which the ATP-dependent dimerization of MinD affects the conformation of the C-terminal region, a potential amphipathic helix, triggering membrane binding.

Recruitment of MinC, an inhibitor of Z-ring formation, to the membrane in Escherichia coli: role of MinD and MinE.

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.

Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE.

Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes. Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles. It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation.