Members of the Bacillus
genus are ubiquitous in nature due to their ability to form spores that are resistant to adverse conditions. The Gulf of Mexico Bacillus
isolates displayed a range of spore production frequencies when sporulation was induced by artificial means during exponential growth. The isolates that had significant levels of sporulation with decoyinine treatment indicate that this chemical was effective in inhibiting GMP synthesis in these isolates. Strains B14850 and B14907 are interesting because of their high levels of sporulation with both the control and decoyinine treatments after 24 h. This could be due to poorly controlled sporulation initiation caused by a mutation resulting in repression of catabolite sensing (6
). Alternatively, these isolates may have salvage pathways that allow them to obtain GMP by other means, so that decoyinine treatment is ineffective in inducing sporulation (40
). With the exception of B14907, the five isolates that did not produce spores during chemical or heat treatment had frequencies of sporulation below 1% under both control and treatment conditions. This indicates that these isolates may have tightly regulated initiation of sporulation that prevents any sporulation occurring when nutrients are present. Further work is needed to elucidate the signals that control sporulation in these isolates.
Eight (66%) of the isolates may have contained temperate phages, since virus-like particles were inducible upon mitomycin C addition. The high incidence of lysogeny in this isolate collection is not unexpected given the prevalence of inducible prophages in the Bacillus
). Based on the absence of lytic activity during the cross-infection experiments with induced lysates, the phages from these isolates were either host specific or already lysogenized with a homoimmune prophage. It is also possible (but relatively rare) for phages to lysogenize a host without producing a lytic infection. Isolate B14912 may have a pseudotemperate phage, since the prophage was not inducible with mitomycin C but the strain displayed a high level of spontaneous prophage induction (50
). Either the three Bacillus
isolates that did not produce significant levels of phage do not contain a temperate phage or the prophages are not mitomycin C inducible. The absence of prophage has been confirmed from in silico
investigation of the draft genome of B14911 (GenBank accession number AAOX00000000). The range of prophage induction in these Bacillus
isolates indicates that the phages in these Bacillus
isolates have different relationships with their hosts.
Isolates B14905, B14906, B14910, and B14850 had significant prophage induction and high levels of sporulation (Fig. ; Table ). It is possible that these isolates may contain temperate phages that enhance host sporulation, such as temperate phages PMB12, SP10, and Φ3226 (46
). Prophage-encoded transcriptional factors may be the mechanism for sporulation enhancement in our Bacillus
isolates, as was seen for Clostridium
phage Φ3226 (53
The lysogen B14905, which produced high levels of spores under decoyinine induction, was selected for sequencing in order to investigate the influence of prophage on sporulation. Sequencing of a mitomycin C-induced lysate resulted in a single phage genome, ΦB05-1. Parallel sequencing of the B14905 chromosomal DNA confirmed the presence of ΦB05-1 as well as three additional prophage-like regions. Genomic analysis of these four regions along with transmission electron microscopy and PCR analysis was performed in order to elucidate which of these regions were inducible prophages.
The genome sizes of the four prophages, ranging from 17,991 bp to 25,898 bp, were much smaller than the sizes of typical tailed prophages (1
). Smaller phage genome sizes are usually associated with lytic phages, defective phages, or phage remnants (26
). However, a survey of the genomes of 113 marine bacterial isolates indicated that most prophage regions were under 30 kb (37
). Gene content, although it can be related to size, is a more important consideration for determining prophages. The genomes of ΦB05-1, ΦB05-3, and ΦB05-4 contained integrases and phage repressor proteins, while that of ΦB05-2 contained only phage replication, terminase, and transposase genes (Fig. ). Since ΦB05-2 was not amplified by PCR from the mitomycin C-induced phage DNA, we believe this region to be a prophage remnant. Prophage remnants, which can contain functional genes, are common in bacterial genomes and are believed to be a result of decay processes (11
ΦB05-4 was the only putative prophage of B14905 that contained several identifiable structural genes. These ORFs were similar to tail genes from the temperate myoviruses from Gram-positive bacteria,
C2 and EJ-1, and to those of the B. subtilis
defective phage PBSX. The presence of lysogeny-like genes, along with the absence of any replicative or packaging genes, on ΦB05-4 implies that this segment may encode a defective phage. Defective phages can be mitomycin C inducible, and some are able to form phage particles that have bactericidal activity but are noninfectious (17
). In the case of PBSX, random 13-kb portions of the host chromosomal genome are packaged but are not injected into other host cells (3
). In this respect, ΦB05-4 may be similar to PBSX since it did not produce a PCR amplicon in the induced phage DNA. A notable difference between the two phages is the lack of identifiable terminase, capsid, and packaging proteins in ΦB05-4. In PBSX, this group of genes, which is about 6,000 bp in length, is located between the xre
repressor and tail genes (23
); this region is not seen in the gene map of ΦB05-4 (Fig. ). Given the absence of injectable phage DNA and the lack of capsid proteins, ΦB05-4 may be a tail-like bacteriocin. Bacteriocins are usually proteinaceous particles that have bactericidal activity toward closely related strains (13
). Some high-molecular-weight bacteriocins resemble phage tails, including the F- and R-type pyocins of Pseudomonas aeruginosa
, carotovoricin Er from Erwinia carotovora
, and a bacteriocin from Bacillus azotofixans
). When B14905 mitomycin C-induced lysates were examined by TEM, many myovirus-like tail particles were observed (Fig. ). The tail tape measure and tail sheath proteins were also expressed in a phage lysate (Fig. ). Based on the evidence from our experiments, we believe that ΦB05-4 is a tail-like bacteriocin, although additional experiments to identify in situ
production of tail proteins in an induced lysate are needed to confirm this.
ΦB05-1 and ΦB05-3 are inducible temperate phages of isolate B14905, based on their amplification from induced phage DNA. Given the lack of identifiable structural genes in these two prophages, it was not possible to identify these phages with the tailed phage structures observed by TEM examination of the lysates. Besides the presence of a lysogeny module, the genomic architectures of these two prophages differ substantially. The genome of ΦB05-1 is smaller and does not have the functional modules that are associated with tailed prophages. An interesting feature of the ΦB05-1 phage genome is the presence of four transcriptional regulators. The one encoded by ORF 2 was not similar to phage-encoded gene products but to the Xre-like DNA binding domains in other Bacillus
transition-state regulators. These transcriptional regulators, which include AbrB and SinR, redirect cellular metabolic activity to use the available source and regulate the expression of sporulation genes during the beginning of the stationary phase (43
). SinR specifically inhibits transcription of spo0A
and stage II sporulation genes until sufficient levels of phosphorylated Spo0A accumulate. Possession of transition-state regulators by prophages may provide an additional level of sporulation control in marine Bacillus
isolates. The lack of similarity of the N terminus of ORF 2 to any known sequences may indicate that this gene is a novel transcriptional regulator. Further experiments are needed to assess the function of this potential prophage-encoded transcriptional regulator in B14905 sporulation.
ORF 17 is another transcriptional regulator found on the genome of ΦB05-1. This gene is located upstream from two putative choloylglycine hydrolase genes; all three genes are similar to those found in two B. cereus
). Choloylglycine hydrolases are bacterial proteins that degrade bile salts in the mammalian intestine (5
). Since B. cereus
is an opportunistic intestinal pathogen, these genes might serve as a survival mechanism for this bacterium. The presence of the genes in an inducible prophage of a marine Bacillus
isolate might be indicative of a horizontal gene transfer event mediated by transduction.
The genome of ΦB05-1 also contained two other ORFs that had weak hits to transcriptional regulators found in bacterial genomes. It is possible that ORF 3 is involved in lytic gene expression, given that its location and orientation are similar to those of cro repressors in other temperate phages (9
). Given its similarity to experimentally characterized protein LlaI, ORF 20 may be involved in phage-encoded resistance. The location of this ORF in the genome is similar to those of methyltransferase proteins found in lytic DNA modification modules encoded by other temperate phages, such as VHML and N15 (34
). The variety of transcriptional regulators in ΦB05-1 may be indicative of the role of these proteins in regulating host and phage functions. For example, Chen et al. (12
) clearly showed that the c
I repressor of coliphage λ not only bound to the operator regions of the lytic λ genes but also could bind to the operators of host metabolic genes. There were multiple c
I binding sites in the operator of the host gene pckA
(phosphoenol carboxykinase), the first gene involved in gluconeogenesis. Downregulating metabolically expensive or wasteful pathways could provide lysogens an advantage during starvation survival, as occurs in most of the oligotrophic oceans (37
The genome of ΦB05-3 is the largest of the prophage-like regions of B14905 and is the closest in genome content to that of a classic tailed phage (11
). Genes involved in phage replication, phage particle assembly, and lysis are similar to those found on temperate phages. Even though the genomic and PCR evidence suggests that ΦB05-3 is an inducible prophage, the genome of this phage was not recovered by sequencing of an induced phage lysate as ΦB05-1 was. It is possible that this phage was induced at a low copy number relative to that of ΦB05-1, so that it was at too low of a concentration to be sequenced. Further functional studies are needed to determine the relationship this phage has with the host strain.
The combination of in silico
analysis of prophage in the B14905 genome and in vivo
molecular studies of the induced phage lysate provided information about host-phage interactions that would not be possible using either approach alone. The results from this study indicate that polylysogeny occurs in marine Bacillus
strains, as has been observed for terrestrial strains (17
). The diversity of the two inducible prophage and two prophage-like elements found on this single bacterial genome supports metagenomic studies that have determined that the diversity of phages in the ocean is vast (4
). This work provides further suggestive evidence that prophages and hosts have coevolved advantageous adaptations to survive under adverse conditions.