The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

Abstract

The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the Escherichia coli host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene motB, located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to E. coli, resulting in cell death or growth arrest depending on the strain and that the presence of motB increases T4 burst size 2-fold. Previous work suggested that motB affects middle gene expression, but our transcriptome analyses of T4 motB^am vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified [(glucosylated, hydroxymethylated-5 cytosine, (GHme-C)] DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.

Keywords: DNA-binding protein; H-NS; MotB; RNA-seq; bacteriophage T4; bacteriostatic; host takeover; transcriptome analysis.

Figure 1

MotB is toxic to E. coli. Heterologous expression of motB in (a) BL21(DE3) or (b) TOP10F’ E. coli causes cell lysis or inhibits growth, respectively. Cell lines containing either the empty vector pNW129 (circles) or expression vector pNW129-MotB (squares) were induced with 0.2% (w/v) arabinose (final concentration) at an OD₆₀₀ between 0.3 and 0.4. Growth curves are representative of three independent replicates. To monitor the amount of MotB produced, cell aliquots at 0, 60 and 90 min post-induction were analyzed by SDS-PAGE. The protein gel slice containing MotB is shown for a representative experiment with the band corresponding to MotB designated by a black arrow. It should be noted that although MotB production is seen at 60 min for BL21(DE3)/pNW129-MotB, no additional accumulation is apparent at 90 min perhaps because of the significant cell death at this time point.

Figure 2

MotB copurifies with host DNA when heterologously produced in E. coli. Lane 1 shows an SDS-PAGE gel, stained with Coomassie, indicating the MotB protein used for the nucleic acid extraction. The agarose gel on the right shows copurifying nucleic acid (lane 3) isolated after phenol-chloroform extraction/ethanol precipitation of affinity-purified MotB and then treated with either RNase-free DNase I (lane 4) or DNase-free RNase A (lane 5). Lane 2 contains GeneRuler DNA marker (Invitrogen). Results are representative of two independent samples.

Figure 3

MotB binds ds and ssDNA. Representative EMSAs for MotB and MotB-His binding to (a) 37 bp P8 oligonucleotide, which contains the T4 Pl₈ promoter from −29 to +8 (dsP8); (b) the top strand of the P8 oligonucleotide (ssP8); (c) a 40 bp fragment containing the E. coli proU operon from +8 to +47 (ProU-40); and (d) a random 32 bp oligonucleotide (Oligo-32). DNA (1 nM) was incubated with the indicated concentrations of MotB or MotB-His at 37 °C for 10 min before electrophoresis on a native polyacrylamide gel. The corresponding K_d(app) is shown below each gel along with the DNA sequence of the top strand. Three independent replicates were performed for each oligonucleotide, except for ProU-40, which was done once with MotB and once with MotB-His.

Figure 4

DNase I footprint of MotB on Pl₈ promoter DNA. The 5’-³²P labeled DNA surrounding the Pl₈ promoter (positions −143 to +75; 0.05 pmol DNA (10.9 pmol total bp), top labeled) was incubated with the indicated amount of MotB-His and treated with DNase I before electrophoresis on a 5% (w/v) polyacrylamide, 7 M urea denaturing gel. Footprints in (a,b) contain the same samples; however, for (b) samples were electrophoresed longer to better resolve the downstream region of the DNA. GA lanes represent G + A ladder. To the left of each gel, a schematic of the Pl₈ promoter region is shown with the T4 late promoter TATA box depicted in yellow and the +1-transcriptional start site indicated by the circular arrow head. Footprints are representative of two independent replicates. Nontemplate sequence (c) of the Pl₈ promoter (−143 to +75) with the TATA box highlighted in yellow, the 37 bp P8 oligo used for gel shift assays indicated by the dashed box, the +1-transcriptional start site indicated by the black arrow and larger font, and the +28 position indicated by the red arrow.

Figure 5

MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.

Figure 6

MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.

Figure 7

MotB improves the burst size of a T4 infection, but has no obvious effect on plaque size. (a) A motB knock-down (T4 motB^am, red) reduces burst size approximately twofold compared to a wild-type infection (T4 wt, grey). Top panel shows burst size as determined by plaque forming units (pfu) over the time course of a T4 infection for a representative set of infections. Bottom panel shows a bar graph of the average burst size with standard deviations determined for three independent replicates. The asterisk (*) indicates significance as determined by an unpaired t-test with a p value ≤ 0.05. (b) A motB knock down (motB^am) does not affect plaque size. Representative image of plaques formed using T4 wt (top panel) or T4 motB^am (bottom panel). Images are representative of at least three independent replicates.

Figure 8

Primer extension products from selected T4 late promoters are similar in T4 wt or a T4 motB^am infection at 10 min post-infection. Histograms show the level of primer extension product obtained from the indicated late promoter using RNA from a T4 motB^am infection (open bar) or a T4 wt (black bar) infection; representative slices of DNA gels are shown below. Primer extensions were repeated using 2 biological replicates. The relative level of product for each replicate is shown as an open square (T4 motB^am). The dotted line indicates the threshold for a twofold change in the level of RNA.