Lysogeny and sporulation in Bacillus isolates from the Gulf of Mexico

Abstract

Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean.

FIG. 1.

Frequency of sporulation in Bacillus isolates after 24 h of decoyinine incubation. The mean percent sporulation for each isolate was used to chart the frequency of sporulation in decoyinine-treated (light-gray bars) and control (dark-gray bars) cultures. Asterisks denote significantly different frequencies of sporulation between the two treatments (n = 3; P < 0.05). Standard deviations are represented by error bars.

FIG. 2.

Prophage production in Bacillus isolates after 24 h of incubation with mitomycin C. The mean prophage production for each isolate in control (dark-gray bars) and mitomycin C-treated (light-gray bars) cultures is shown. Asterisks denote significantly different levels of prophage production (P < 0.05). Standard deviations are represented by error bars.

FIG. 3.

Prophage production and sporulation in Bacillus isolates. For each strain, the percent difference in phage production (mitomycin C treated versus spontaneous) and the percent difference in number of spores, as outlined in Table 3, were graphed.

FIG. 4.

Genomic map of B14905 prophage-like regions. KODON was used to construct the gene map. Patterns were assigned based on putative ORF function (Table 4).

FIG. 5.

Agarose gel electrophoresis of B14905 DNA PCR amplicons. Lanes 1 and 12, Fisher 100-bp DNA standard; lanes 2 through 6, host chromosomal DNA (lane 2, ΦB05-1 primer set; lane 3, ΦB05-2 primer set; lane 4, ΦB05-3 primer set; lane 5, ΦB05-4 primer set; lane 6, host primer set); lanes 7 through 11, CsCl phage lysate DNA (lane 7, ΦB05-1 primer set; lane 8, ΦB05-2 primer set; lane 9, ΦB05-3 primer set; lane 10, ΦB05-4 primer set; lane 11, host primer set). Values at left are sizes in bp. Primer sequences are in given in Table 2.

FIG. 6.

SDS-PAGE of proteins associated with B14905 lysates. The molecular mass standard is shown in lanes 2 and 4, with sizes in kilodaltons given on the left. Lane 1, CsCl-purified preparation; lane 5, polyethylene glycol-precipitated preparation.

FIG. 7.

TEM micrographs of B14905 induced phage lysates. (A, B, E, and F) Magnification, ×100,000. Bar, 100 nm. (C and D) Magnification, ×150,000. Bar, 50 nm.