(PY79)

(PY79). homologs of Hfq: Hfq1 and Mirabegron Hfq2 from the chromosome, and Hfq3 from the pXO1 virulence plasmid. Results In this study, we utilized overexpression as a strategy to examine the impact of Hfq3 on physiology. The increase in Hfq3 protein levels led to anomalous cell shape and chain formation, which manifested as a severe growth defect. This phenotype was specific to at similar levels was not toxic. Toxicity was dependent on residues on the distal face of Hfq3 that are involved in mRNA binding in other bacterial species. Conclusions Thus, we hypothesize that Hfq3 interacts with RNA(s) involved in essential functions in the cell, leading to increased binding upon overexpression that either sequesters or accelerates degradation of RNAs important for growth. These results not only aid in elucidating the role of Hfq proteins in from has no major effects on physiology, and sRNA regulation is not impacted [11]. Similarly, while Hfq associates with numerous sRNAs [12], strains exhibit only subtle effects on their physiology and transcriptome [13, 14]. In contrast, in identified multiple phenotypes associated with the knockdown of expression, including the first evidence of an within its genome. Two copies are located on the chromosome (genes for Hfq1 and Hfq2), and the third (gene for Hfq3) is encoded by the pXO1 virulence plasmid, which also produces the bacteriums toxin components. Hfq2 is closest in protein sequence to the single copy of Hfq in the close relative Hfq (Fig.?1). Each of the three genes is expressed at the mRNA level during the mid-log phase of growth [18]. Open in a separate window Fig. 1 Sequence Alignment of Hfq Proteins. Alignment of Hfq, Hfq, Hfq, and Hfq1, Hfq2 and Hfq3 protein sequences using Clustal Omega [43]. BOXSHADE was used to create the image; black shading indicates identical residues, and grey shading Mirabegron denotes biochemically similar residues. The location of the Sm2 RNA binding motif is labeled Our recent work [18] and that of Panda genus Mirabegron by characterization of the structure and function of recombinant Hfq proteins. Hfq1 (BA_1656; Hfq2 in [19]) does not form the typical hexameric structure expected Rabbit Polyclonal to Actin-pan for the protein family; instead, it behaves as a monomer that appears to disrupt Hfqs chaperone activity with a dominant negative phenotype [18]. In contrast, Hfq2 (BA_3842; Hfq1 in [19]) forms the standard hexamer [18]. Purified Hfq3 (GBAA_RS29265) behaves as a mixture of hexamer and monomer, and His-Hfq3 partially complements the phenotype [18]. Panda Hfq proteins, as well as its ability to form hexamers and its presence on one of strains were used for cloning. strain Ames 35 (pXO1+, pXO2?; derived from [20]) and strain PY79 [21] were grown in LB broth/plate or NBY broth/plate (containing 0.8% nutrient broth, 0.3% yeast extract, and 0.9% sodium bicarbonate). LB and NBY/0.9% sodium bicarbonate cultures were incubated at 37?C in air or a CO2 environment, respectively. Hfq complementation strains (YN585 derivatives) were grown on MacConkey agar plates (BD Difco; Franklin Lakes, NJ) incubated at 37?C in air. When appropriate, antibiotics were added to both liquid and solid media: ampicillin (100?g/mL); kanamycin (15?g/mL). Cloning and transformation Each gene was cloned into the pSW4 shuttle expression vector [22] using the NdeI and BamHI restriction sites. After passage through SCS110 to prepare unmethylated DNA, pSW4 vectors were transformed into via electroporation. pSW4 expression vectors were transformed into strain PY79 via natural competency using Modified Competency Media (3?mM sodium citrate, 2% glucose, 20?g/mL ferric ammonium citrate, 0.1% casein hydrolysate, 0.2% potassium glutamate, 100?mM potassium phosphate, 10?mM MgSO4) [23]. Total lysate and western blot analysis Bacteria were grown in LB or NBY/0.9% sodium bicarbonate cultures in either air (LB) or 15% CO2 (NBY). Cells were harvested via centrifugation, and cell pellets were lysed in 1X PBS via beadbeating. Protein concentrations of lysate supernates were estimated via an A280 measurement, and gels/blots were normalized by loading equal amounts of total protein per well, which was validated by Coomassie Blue staining. Proteins were separated on 4C20% Tris-glycine gels (Life Technologies; western blot) or 16% Tricine gels (Life Technologies; total lysate). Samples were run as semi-native (not heated) vs. boiled (5?min, 99?C), and were prepared with either 2X Tris-glycine SDS loading buffer (Life Technologies; final [SDS] 1%) or 2X Tricine SDS loading buffer (Life Technologies; final [SDS] 4%). Both gel systems utilized running Mirabegron buffer containing 0.1% SDS. Following SDS-PAGE, 16% Tricine gels were Coomassie.