CRISPR-Cas systems in the marine actinomycete Salinispora: linkages with phage defense,microdiversity and biogeography

Background

Prokaryotic CRISPR-Cas systems confer resistance to viral infection and thus mediate bacteria-phage interactions. However, the distribution and functional diversity of CRISPRs among environmental bacteria remains largely unknown. Here, comparative genomics of 75 Salinispora strains provided insight into the diversity and distribution of CRISPR-Cas systems in a cosmopolitan marine actinomycete genus.

Results

CRISPRs were found in all Salinispora strains, with the majority containing multiple loci and different Cas array subtypes. Of the six subtypes identified, three have not been previously described. A lower prophage frequency in S. arenicola was associated with a higher fraction of spacers matching Salinispora prophages compared to S. tropica, suggesting differing defensive capacities between Salinispora species. The occurrence of related prophages in strains from distant locations, as well as spacers matching those prophages inserted throughout spacer arrays, indicate recurring encounters with widely distributed phages over time. Linkages of CRISPR features with Salinispora microdiversity pointed to subclade-specific contacts with mobile genetic elements (MGEs). This included lineage-specific spacer deletions or insertions, which may reflect weak selective pressures to maintain immunity or distinct temporal interactions with MGEs, respectively. Biogeographic patterns in spacer and prophage distributions support the concept that Salinispora spp. encounter localized MGEs. Moreover, the presence of spacers matching housekeeping genes suggests that CRISPRs may have functions outside of viral defense.

Conclusions

This study provides a comprehensive examination of CRISPR-Cas systems in a broadly distributed group of environmental bacteria. The ubiquity and diversity of CRISPRs in Salinispora suggests that CRISPR-mediated interactions with MGEs represent a major force in the ecology and evolution of this cosmopolitan marine actinomycete genus.

Electronic supplementary material

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Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs

Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptive defence system that provides resistance against alien replicons such as viruses and plasmids. Spacers in a CRISPR cassette confer immunity against viruses and plasmids containing regions complementary to the spacers and hence they retain a footprint of interactions between prokaryotes and their viruses in individual strains and ecosystems. The human gut is a rich habitat populated by numerous microorganisms, but a large fraction of these are unculturable and little is known about them in general and their CRISPR systems in particular.

Results

We used human gut metagenomic data from three open projects in order to characterize the composition and dynamics of CRISPR cassettes in the human-associated microbiota. Applying available CRISPR-identification algorithms and a previously designed filtering procedure to the assembled human gut metagenomic contigs, we found 388 CRISPR cassettes, 373 of which had repeats not observed previously in complete genomes or other datasets. Only 171 of 3,545 identified spacers were coupled with protospacers from the human gut metagenomic contigs. The number of matches to GenBank sequences was negligible, providing protospacers for 26 spacers.Reconstruction of CRISPR cassettes allowed us to track the dynamics of spacer content. In agreement with other published observations we show that spacers shared by different cassettes (and hence likely older ones) tend to the trailer ends, whereas spacers with matches in the metagenomes are distributed unevenly across cassettes, demonstrating a preference to form clusters closer to the active end of a CRISPR cassette, adjacent to the leader, and hence suggesting dynamical interactions between prokaryotes and viruses in the human gut. Remarkably, spacers match protospacers in the metagenome of the same individual with frequency comparable to a random control, but may match protospacers from metagenomes of other individuals.

Conclusions

The analysis of assembled contigs is complementary to the approach based on the analysis of original reads and hence provides additional data about composition and evolution of CRISPR cassettes, revealing the dynamics of CRISPR-phage interactions in metagenomes.

Electronic supplementary material

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Motif depletion in bacteriophages infecting hosts with CRISPR systems

Background

CRISPR is a microbial immune system likely to be involved in host-parasite coevolution. It functions using target sequences encoded by the bacterial genome, which interfere with invading nucleic acids using a homology-dependent system. The system also requires protospacer associated motifs (PAMs), short motifs close to the target sequence that are required for interference in CRISPR types I and II. Here, we investigate whether PAMs are depleted in phage genomes due to selection pressure to escape recognition.

Results

To this end, we analyzed two data sets. Phages infecting all bacterial hosts were analyzed first, followed by a detailed analysis of phages infecting the genus Streptococcus, where PAMs are best understood. We use two different measures of motif underrepresentation that control for codon bias and the frequency of submotifs. We compare phages infecting species with a particular CRISPR type to those infecting species without that type. Since only known PAMs were investigated, the analysis is restricted to CRISPR types I-C and I-E and in Streptococcus to types I-C and II. We found evidence for PAM depletion in Streptococcus phages infecting hosts with CRISPR type I-C, in Vibrio phages infecting hosts with CRISPR type I-E and in Streptococcus thermopilus phages infecting hosts with type II-A, known as CRISPR3.

Conclusions

The observed motif depletion in phages with hosts having CRISPR can be attributed to selection rather than to mutational bias, as mutational bias should affect the phages of all hosts. This observation implies that the CRISPR system has been efficient in the groups discussed here.

Electronic supplementary material

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CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

Background

Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders'' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers'' targets.

Results

We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs.

Conclusions

We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.     

Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses

Background

Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases.

Methodology and Principal Findings

Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales).

Conclusions and Significance

We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.     

CRISPR analysis of bacteriophage‐insensitive mutants (BIMs) of industrial Streptococcus thermophilus– implications for starter design

Aims: An efficient approach for generation of bacteriophage‐insensitive mutants (BIMs) of Streptococcus thermophilus starters was described in our laboratory [Mills et al. (2007) J Microbiol Methods 70 , 159–164]. The aim of this study was to analyse the phage resistance mechanism responsible for BIM formation. Methods and Results: Three clustered regularly interspaced short palindromic repeat (CRISPR) regions have been identified in Strep. thermophilus, and Strep. thermophilus can integrate novel spacers into these loci in response to phage attack. Characterization of three sets of BIMs indicated that two sets had altered CRISPR1 and/or CRISPR3 loci. A range of BIMs of yoghurt starter CSK938 were generated with the same phage in different phage challenge experiments, and each acquired unique spacer regions ranging between one and four new spacers in CRISPR1. In addition, the BIM that acquired only one new spacer in CRISPR1 also acquired an additional spacer in CRISPR3. A fourth BIM, generated with a different phage, had two spacers deleted from CRISPR1 but acquired two spacers in CRISPR3. Analysis of the Mozzarella starter CSK939 and its associated BIMs indicated that formation of second generation BIMs does not lead to increases in spacer number but to alterations in spacer regions. BIMs of an exopolysaccharide (EPS)‐producing strain that lost the ability to produce EPS did not harbour an altered CRISPR, suggesting that phage sensitivity may be related to the EPS‐producing phenotype. Conclusions: Acquisition/deletion of new spacers in CRISPR loci in response to phage attack generates distinctly individual variants. It also demonstrates that other modifications may be responsible for the phage resistance of Strep. thermophilus BIMs. Significance and Impact of the Study: Isolation of individual BIMs that have unique spacers towards the leader region of the CRISPR locus may be a very useful approach for rotation strategies with the same starter backbone. Upon phage infection, BIMs ‘in reserve’ can be slotted into the rotation scheme.     

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

Background

In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.

Results

Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.

Conclusions

The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Electronic supplementary material

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Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow

Background

Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes.

Results

Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE.

Conclusions

The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

Electronic supplementary material

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Insight into microevolution of Yersinia pestis by clustered regularly interspaced short palindromic repeats

Background

Yersinia pestis, the pathogen of plague, has greatly influenced human history on a global scale. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), an element participating in immunity against phages'' invasion, is composed of short repeated sequences separated by unique spacers and provides the basis of the spoligotyping technology. In the present research, three CRISPR loci were analyzed in 125 strains of Y. pestis from 26 natural plague foci of China, the former Soviet Union and Mongolia were analyzed, for validating CRISPR-based genotyping method and better understanding adaptive microevolution of Y. pestis.

Methodology/Principal Findings

Using PCR amplification, sequencing and online data processing, a high degree of genetic diversity was revealed in all three CRISPR elements. The distribution of spacers and their arrays in Y. pestis strains is strongly region and focus-specific, allowing the construction of a hypothetic evolutionary model of Y. pestis. This model suggests transmission route of microtus strains that encircled Takla Makan Desert and ZhunGer Basin. Starting from Tadjikistan, one branch passed through the Kunlun Mountains, and moved to the Qinghai-Tibet Plateau. Another branch went north via the Pamirs Plateau, the Tianshan Mountains, the Altai Mountains and the Inner Mongolian Plateau. Other Y. pestis lineages might be originated from certain areas along those routes.

Conclusions/significance

CRISPR can provide important information for genotyping and evolutionary research of bacteria, which will help to trace the source of outbreaks. The resulting data will make possible the development of very low cost and high-resolution assays for the systematic typing of any new isolate.     

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages

Background

Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution.

Results

Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named “core genome”, and the second group, containing mostly short ORFs without assigned functions was called “accessory genome”. Like in other phage groups, variable genes are confined to specific regions in the genome.

Conclusion

Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

Electronic supplementary material

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Genetics of CRISPR arrays in <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium 14028 associated with foreign DNA decay

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a genetic locus of prokaryotes and contain highly conserved direct repeats, spacers, and CRISPR-associated genes. Spacers in CRISPRs are known as adaptive immune markers and reveal what types of phage or foreign DNA have been introduced in the past. The primary objective of this study was to analyze spacer sequences in CRISPR arrays of 15 Salmonella enterica subspecies and to determine if Salmonella CRISPRs are indeed involved in resistance to foreign DNAs. Using a bioinformatics algorithm, the CRISPR arrays of 15 subspecies of S. enterica were predicted. The transformation efficiencies of the wild-type and mutant strains lacking a space were determined using the plasmid harboring the same sequences with the space. Analysis of the CRISPR arrays indicated that S. Typhimurium encoded three possible CRISPR regions in the genome. Notably, 48 or 55 spacers were predicted in the genomes of S. Typhimurium 14028 and LT2 strains, respectively, and 39 were precisely identical. To confirm this prediction, the predicted CRISPR regions of S. Typhimurium 14028 were sequenced using the specific primers. Interestingly, a homology search of individual spacers found that the 2nd spacer of CRISPR 2 was nearly identical to a partial genome region of phage FSL SP-016. The mutant strain showed two to threefold increased transformation efficiency compared to that of the wild-type strain. These results demonstrate that the spacer sequence is dependent on genetic relations, especially for adaptive immunity against phage or foreign DNAs.     

Chemical synthesis of bacteriophage G4

Background

Due to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity.

Methodology/Principal Findings

Full-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes.

Conclusions

Our results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application.     

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging

Background

Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.

Results

Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.

Conclusions

The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

Electronic supplementary material

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Bioinformatics analyses of <Emphasis Type="Italic">Shigella</Emphasis> CRISPR structure and spacer classification

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of a variety of archaea and bacteria and indicative of the bacterial ecological adaptation, conferring acquired immunity against invading foreign nucleic acids. Shigella is an important pathogen for anthroponosis. This study aimed to analyze the features of Shigella CRISPR structure and classify the spacers through bioinformatics approach. Among 107 Shigella, 434 CRISPR structure loci were identified with two to seven loci in different strains. CRISPR-Q1, CRISPR-Q4 and CRISPR-Q5 were widely distributed in Shigella strains. Comparison of the first and last repeats of CRISPR1, CRISPR2 and CRISPR3 revealed several base variants and different stem-loop structures. A total of 259 cas genes were found among these 107 Shigella strains. The cas gene deletions were discovered in 88 strains. However, there is one strain that does not contain cas gene. Intact clusters of cas genes were found in 19 strains. From comprehensive analysis of sequence signature and BLAST and CRISPRTarget score, the 708 spacers were classified into three subtypes: Type I, Type II and Type III. Of them, Type I spacer referred to those linked with one gene segment, Type II spacer linked with two or more different gene segments, and Type III spacer undefined. This study examined the diversity of CRISPR/cas system in Shigella strains, demonstrated the main features of CRISPR structure and spacer classification, which provided critical information for elucidation of the mechanisms of spacer formation and exploration of the role the spacers play in the function of the CRISPR/cas system.     

Multispacer sequence typing relapsing fever Borreliae in Africa

Background

In Africa, relapsing fevers are neglected arthropod-borne infections caused by closely related Borrelia species. They cause mild to deadly undifferentiated fever particularly severe in pregnant women. Lack of a tool to genotype these Borrelia organisms limits knowledge regarding their reservoirs and their epidemiology.

Methodology/Principal Findings

Genome sequence analysis of Borrelia crocidurae, Borrelia duttonii and Borrelia recurrentis yielded 5 intergenic spacers scattered between 10 chromosomal genes that were incorporated into a multispacer sequence typing (MST) approach. Sequencing these spacers directly from human blood specimens previously found to be infected by B. recurrentis (30 specimens), B. duttonii (17 specimens) and B. crocidurae (13 specimens) resolved these 60 strains and the 3 type strains into 13 species-specific spacer types in the presence of negative controls. B. crocidurae comprised of 8 spacer types, B. duttonii of 3 spacer types and B. recurrentis of 2 spacer types.

Conclusions/Significance

Phylogenetic analyses of MST data suggested that B. duttonii, B. crocidurae and B. recurrentis are variants of a unique ancestral Borrelia species. MST proved to be a suitable approach for identifying and genotyping relapsing fever borreliae in Africa. It could be applied to both vectors and clinical specimens.     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage,Sf101, from a serotype 7a strain

Background

Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of Rha^III, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.

Results

In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.

Conclusions

This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.

Electronic supplementary material

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