Variovorax paradoxus is a microorganism of special interest due to its diverse metabolic capabilities, including the biodegradation of both biogenic compounds and anthropogenic contaminants. V. paradoxus also engages in mutually beneficial interactions with both bacteria and plants. The complete genome sequence of V. paradoxus S110 is composed of 6,754,997 bp with 6,279 predicted protein-coding sequences within two circular chromosomes. Genomic analysis has revealed multiple metabolic features for autotrophic and heterotrophic lifestyles. These metabolic diversities enable independent survival, as well as a symbiotic lifestyle. Consequently, S110 appears to have evolved into a superbly adaptable microorganism that is able to survive in ever-changing environmental conditions. Based on our findings, we suggest V. paradoxus S110 as a potential candidate for agrobiotechnological applications, such as biofertilizer and biopesticide. Because it has many associations with other biota, it is also suited to serve as an additional model system for studies of microbe-plant and microbe-microbe interactions.
We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules." We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules." toxic shock syndrome staphylococcal pathogenicity island toxic shock syndrome toxin 1 open reading frame Todd-Hewitt staphylococcal enterotoxin Staphylococcus aureus is a leading etiologic agent of both nosocomial and community-acquired infections worldwide. These infections range from fairly benign cutaneous infections, such as furuncles, to potentially fatal diseases, including endocarditis and toxic shock syndrome (TSS)1(reviewed in Ref. 1.Tenover F.C. Gaynes R.P. Fischetti V.A. Novick R.P. Ferretti J.J. Portnoy D.A. Rood J.I. Gram-positive Pathogens. ASM Press, Washington, D. C.2000: 414-421Google Scholar). Its ability to cause this range of disease is due in part to its elaboration of a vast array of both cell surface-associated and secreted virulence factors. Among the secreted factors are the pyrogenic toxin superantigens that have the ability to activate large populations (10–50%) of T lymphocytes in a manner specific to the variable region of the β-chain of the T-cell receptor (2.Marrack P. Kappler J. Science. 1990; 248: 705-711Crossref PubMed Scopus (1210) Google Scholar). The ensuing massive cytokine release results in the symptoms of TSS, including fever, hypotension, rash, vomiting, diarrhea, multiple organ failure, disseminated intravascular coagulation, and desquamation. The staphylococcal enterotoxins (SEs), members of the superantigen family, are associated with both TSS and food poisoning and have proven emetic activities that appear to be separable from their superantigenic activity (3.Hovde C.J. Marr J.C. Hoffmann M.L. Hackett S.P. Chi Y.I. Crum K.K. Stevens D.L. Stauffacher C.V. Bohach G.A. Mol. Microbiol. 1994; 13: 897-909Crossref PubMed Scopus (78) Google Scholar). Most, if not all, staphylococcal superantigens are encoded by accessory genetic elements that are either mobile or appear to have been mobile at one time (reviewed in Ref. 4.McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). These identified elements include plasmids, transposons, prophages, and the pathogenicity islands. The staphylococcal pathogenicity islands (SaPIs), of which five have, until recently, been described (SaPI1–4 and SaPIbov), are the first clearly defined pathogenicity islands in Gram-positive bacteria, and each encodes one or more of the staphylococcal superantigens (reviewed in Ref. 5.Novick R.P. Schlievert P. Ruzin A. Microbes Infect. 2001; 3: 585-594Crossref PubMed Scopus (115) Google Scholar). SEB, SEC, SEK, SEL, SEQ, and toxic shock syndrome toxin 1 (TSST-1) are known to be encoded by one or more of these phage-related elements (reviewed in Ref. 4.McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). More recently, Kuroda et al.(6.Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google Scholar) identified six novel pathogenicity islands in the complete genomes of two S. aureus strains, N315 and Mu50, including three that carried tstH (encoding TSST-1), two that carried clusters of staphylococcal exotoxin-like proteins, and one, present in both strains, that carried clusters of serine proteases and enterotoxins. These genomic loci meet the generally accepted requirements of the pathogenicity island subgroup of "genomic islands" as defined previously (7.Hacker J. Kaper J.B. Annu. Rev. Microbiol. 2000; 54: 641-679Crossref PubMed Scopus (938) Google Scholar, 8.Hentschel U. Hacker J. Microbes Infect. 2001; 3: 545-548Crossref PubMed Scopus (87) Google Scholar). They are present in the genomes of many staphylococci but absent from closely related strains, they are relatively large genomic fragments (>15 kb), they differ in GC content from the rest of the chromosome, they are flanked by direct repeats likely generated upon insertion of the elements into the genome, some are associated with tRNA loci, and they possess genes coding for genetic mobility, including conserved integrases. The prototypical staphylococcal pathogenicity island, SaPI1, was identified and characterized by Lindsay et al. (9.Lindsay J.A. Ruzin A. Ross H.F. Kurepina N. Novick R.P. Mol. Microbiol. 1998; 29: 527-543Crossref PubMed Scopus (317) Google Scholar) as the genetic element encoding TSST-1, the only superantigen to be associated with nearly all cases of menstrual TSS. SaPI1 is 15.2 kb in length, flanked by a 17-nucleotide direct repeat, contains a functional integrase (int) gene, and is located near the tyrB locus in strain RN4282. It also appears to encode a second superantigen, SEK, and part of a third superantigen, SEQ. Mobility of SaPI1 has been demonstrated only in the presence of a helper phage, such as 80α. Ruzin et al. (10.Ruzin A. Lindsay J. Novick R.P. Mol. Microbiol. 2001; 41: 365-377Crossref PubMed Scopus (151) Google Scholar) have demonstrated that SaPI1 appears to parasitize excision, replication, and encapsidation functions of phage 80α in a relationship that is similar to that between coliphages P4 and P2. During the growth of phage 80α, SaPI1 excises from its unique chromosomal insertion site,attc, replicates in the linear form, interferes with phage growth, and is encapsidated into specialized phage heads. Upon transduction to a recipient organism, SaPI1 integrates by the classical Campbell mechanism into the attc site for which the SaPI1-coded integrase is necessary. Because islands with different att sites appear to have dissimilar integrases (6.Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google Scholar), it may well be that the integrase carried by the island determines the integration site in the genome. Existence of these toxin genes on mobile genetic elements implies their transfer between staphylococcal strains as well as other bacterial species by horizontal transfer. Furthermore, these elements are not uniformly distributed among clinical isolates. Thus, these mobile elements likely have played and continue to play an integral role in the evolution of S. aureus as a species and as a pathogen. Indeed, recent evidence supports the hypothesis that virulence traits are spread by horizontal transfer, particularly in nosocomial infections, and that the presence of accessory genetic elements within a strain may affect the acquisition and loss of other mobile genetic elements (11.Moore P.C. Lindsay J.A. J. Clin. Microbiol. 2001; 39: 2760-2767Crossref PubMed Scopus (146) Google Scholar). These islands may also form the basis for toxin gene exclusion. For instance, in testing thousands of strains, our laboratory has never identified a clinical isolate that produced both TSST-1 and SEB. It has been determined that these toxins are encoded by different pathogenicity islands that appear to exclude each other from their respective integration sites. However, despite the identification of numerous pathogenicity islands and their likely importance in the evolution of Staphylococcus as a pathogen, the origin and functions of pathogenicity islands remain areas with little investigation. All of the SaPIs have multiple open reading frames (ORFs), many of which have no identifiable homologs. To this point, the issues of whether or not these ORFs are expressed and whether their function might be in the regulation of island-associated superantigens or only in the maintenance and transfer of the islands have not been addressed. In this study, we report the complete sequence and map of SaPI3 encoding SEB and the more recently identified enterotoxins, SEK (12.Orwin P.M. Leung D.Y. Donahue H.L. Novick R.P. Schlievert P.M. Infect. Immun. 2001; 69: 360-366Crossref PubMed Scopus (177) Google Scholar) and SEQ. 2P. M. Orwin, D. Y. M. Leung, H. L. Donahue, G. A. Bohach, and P. M. Schlievert, submitted for publication.2P. M. Orwin, D. Y. M. Leung, H. L. Donahue, G. A. Bohach, and P. M. Schlievert, submitted for publication.Furthermore, we describe for the first time the expression of the numerous genes contained on a staphylococcal pathogenicity island using DNA microarray technology. We then discuss the implications for the evolution of the staphylococcal pathogenicity islands. COL is a prototypical methicillin-resistant isolate of S. aureus that is currently being sequenced by The Institute for Genomic Research. MN NJ is a methicillin-sensitive isolate of S. aureus from a case of nonmenstrual TSS in which our laboratory has identified two novel superantigens, SEK (12.Orwin P.M. Leung D.Y. Donahue H.L. Novick R.P. Schlievert P.M. Infect. Immun. 2001; 69: 360-366Crossref PubMed Scopus (177) Google Scholar) and SEQ.2S. aureus MN8, also a methicillin-sensitive isolate of S. aureus, was used as a source of genomic template for amplification of virulence gene probes for the DNA microarrays and was isolated before 1980 from a case of menstrual TSS (13.Schlievert P.M. Blomster D.A. J. Infect. Dis. 1983; 147: 236-242Crossref PubMed Scopus (146) Google Scholar). Preliminary sequence data for the S. aureus strain COL was obtained from The Institute for Genomic Research website (www.tigr.org). A primer walking-based approach was used to amplify by PCR and sequence ∼3.4 kb that spanned the unsequenced loci of SaPI3 in the COL genomic sequence data base. Automated sequencing using an ABI model 377 was performed with the assistance of the Advanced Genetic Analysis Center (University of Minnesota, St. Paul, MN). 50-ml cultures of S. aureus MN NJ were grown aerobically with shaking at 37 °C in either Todd-Hewitt (TH) broth (BC PharMingen) or rabbit serum (Invitrogen). Two independent cultures in each medium were grown in parallel, and samples were removed at the exponential, postexponential, and stationary phases of growth (2, 3, and 8 h after inoculation with an initial cell density of A600 nm = 0.1). Expression of SaPI3 ORFs and other virulence-associated genes was quantified using DNA microarrays. For cultures exposed to altered oxygen levels, 1 ml of TH broth was inoculated with S. aureus MN NJ from an overnight culture to an initial cell density of A600 nm = 0.1 in a 35 × 12-mm-diameter polystyrene Petri dish (Nunc, Roskilde, Denmark). All cultures were placed into sealed, humidified Plexiglass cell culture chambers (20 × 26 × 7.5 cm, internal dimensions; Mishell-Dutton) (14.Mishell B.B. Mishell R.I. Mishell B.B. Shiigi S.M. Selected Methods in Cellular Immunology. W. H. Freeman and Company, San Francisco1980: 30-37Google Scholar), flushed with gas mixtures containing either 1% oxygen (v/v) or 21% oxygen (v/v) balanced with nitrogen and 7% carbon dioxide (Praxair, St. Louis, MO), and sealed. Chambers were then incubated at 37 °C with orbital shaking (∼125 rpm). Samples for the exponential, postexponential, and stationary phases of growth were removed at ∼2, 3, and 8 h after inoculation, respectively, and expression of SaPI3 genes was quantified using DNA microarrays. We have previously determined that there is no significant difference in growth parameters (cell densities or timing of entry into the growth phases of interest) between cultures grown in 1% oxygen and 21% oxygen (v/v) balanced with nitrogen and 7% carbon dioxide (15.Yarwood J.M. Schlievert P.M. J. Clin. Microbiol. 2000; 38: 1797-1803Crossref PubMed Google Scholar,16.Yarwood J.M. McCormick J.K. Schlievert P.M. J. Bacteriol. 2001; 183: 1113-1123Crossref PubMed Scopus (235) Google Scholar). Two Dutch-belted rabbits were immunized with SEB, which is made in high concentrations (>10 μg/ml) by MN NJ grown in vitro. Rabbits were immunized by three subcutaneous injections at 2-week intervals, with each injection containing 25 μg of purified SEB resuspended in 0.5 ml of phosphate-buffered saline and emulsified in 0.5 ml of incomplete Freund's adjuvant. Development of antibody to SEB was determined by enzyme-linked immunosorbent assay of serum samples taken 1 week after the final immunization. The two rabbits developed anti-SEB (IgG) titers of 1:5,120 and 1:10,240, respectively, as compared with preimmune titers of <1:20. Sterilized perforated hollow polyethylene golf balls were implanted subcutaneously in four Dutch-belted rabbits (17.Scott D.F. Kling J.M. Kirkland J.J. Best G.K. Infect. Immun. 1983; 39: 383-387Crossref PubMed Google Scholar). Implantation of the polyethylene balls and subsequent healing created transudate-filled cavities in the rabbits with volumes of ∼15 ml that contained few host cells, thus enabling the preparation of staphylococcal RNA relatively free of contaminating host RNA. 6 weeks after implantation of the polyethylene balls, ∼1010 colony-forming units of S. aureus MN NJ grown in TH medium were collected by centrifugation from the late exponential phase of growth (cell density of cultures was 6.7 × 108 colony-forming units/ml), resuspended in 2 ml of phosphate-buffered saline, and injected into the implanted polyethylene balls. Samples were removed from the inoculum culture before centrifugation for use in expression analysis by DNA microarrays. 2 ml of transudate containing S. aureus were then removed from the infection chambers at the indicated times after inoculation using a sterile syringe, S. aureus was enumerated by plating, and expression of SaPI3 genes was quantified using DNA microarrays. Analysis of staphylococcal gene expression in vitro and in vivo using DNA microarrays was performed as described elsewhere (www.agac.umn.edu/microarray/protocols/protocols.htm). In brief, a library of targets representing 68 genes from S. aureus MN NJ and MN8 was constructed with primers designed to amplify fragments of ∼300 bp of each gene from genomic DNA. Two successive rounds of PCR were performed to minimize genomic DNA contamination in the amplification products, and the final 100-μl reactions were checked for quality on agarose gels and purified with the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). The purified products were printed in triplicate using a Total Array System robot (BioRobotics, Boston, MA). Cell pellets from centrifuged samples of S. aureus cultures were flash-frozen in liquid nitrogen. Total RNA was prepared using the RNeasy Mini Kit (Qiagen) according to the manufacturer's directions. DNA was removed from the RNA preparations using the RNase-free DNase Set (Qiagen) according to the manufacturer's directions. cDNA prepared from RNA from S. aureus cultures to be compared was labeled with either Cy3 or Cy5 fluorescent dye (Amersham Biosciences) and competitively hybridized with the printed microarrays. Images of the hybridized arrays were obtained with a Scanarray 5000 microarray scanner (GSI Lumonics, Watertown, MA). One independent hybridization (on triplicate arrays) was conducted for each of two independent experiments. Fluorescence intensities for individual spots were normalized based on the total intensity of fluorescence in the Cy3 and Cy5 channels. Fluorescence intensity was determined as the average intensity of the triplicate spots for each gene. Total fluorescence for each gene was normalized between arrays for independent experiments, the data were combined from both experiments, and statistical significance was determined using Student's t test to compare expression data from the two growth conditions of interest. SaPI3 ORFs were determined as being expressed if the fluorescence intensity was at least twice that of background levels established using negative controls (probes for genes not expressed by strains MN NJ and COL) and if fluorescence was detected in each of the triplicate arrays for each independent experiment. To account for possible bias in labeling of cDNA by either Cy3 or Cy5, dye labeling was reversed in the second independent experiment for each of several experimental conditions. No dye bias was detected. Clustering based on similarity of expression profiles and visualization were performed using the software program Spotfire DecisionSite 6.1 (www.spotfire.com). Similarities between expression profiles of individual genes in all 11 experimental conditions were calculated using the "Euclidean distance" method. Data base searches of the unfinished S. aureus COL genome (www.tigr.org) revealed the presence of large segments homologous to SaPI1. Because COL does not produce TSST-1, we hypothesized that this sequence comprised a novel pathogenicity island, which we termed SaPI3. Using the SaPI1 sequence and SaPI3 partial sequence as guides, primers were designed to complete the sequence of SaPI3 in the COL strain. In all, a PCR and primer walking-based approach was used to sequence ∼3.4 kb of the 15,936-bp SaPI3. SaPI3 was determined to have 24 ORFs potentially encoding proteins over 50 amino acids in length, 3 of which encoded staphylococcal enterotoxin serotypes B, K, and Q, and many of which have homologs in SaPI1 and SaPIbov (Fig. 1; Table I). We were also able to identify the presence of SaPI3 in the clinical isolate S. aureus MN NJ, a known SEB producer, by PCR analysis and sequencing of the same three regions as in strain COL (data not shown). MN NJ is an isolate from a case of nonmenstrual TSS in which our laboratory has described the presence of two novel enterotoxins, SEK (12.Orwin P.M. Leung D.Y. Donahue H.L. Novick R.P. Schlievert P.M. Infect. Immun. 2001; 69: 360-366Crossref PubMed Scopus (177) Google Scholar) and SEQ.2 The repeated 17-nucleotide sequences flanking SaPI3 were identical to the att sites of SaPI1 (5′-TTATTTAGCAGGATAA-3′) and thus might form the basis of pathogenicity island exclusion (i.e. the lack of TSST-1 and SEB in the same clinical isolates). In general, the identified SaPI3 genes form two apparent transcriptional blocks with ORFs 2–18 (including SEB) oriented toward the left of the island, whereas ORFs 19–24 (including SEK and SEQ) are oriented to the right (Fig. 1). The overall GC content of SaPI3 was 31.4%, somewhat lower than the 32.8–32.9% found for the whole genome of S. aureus (6.Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1564) Google Scholar).Table IDescription of SaPI3 ORFsNameLengthPredicted molecular mass1-aDetermined using the Compute pI/Mw tool at ca.expasy.org/tools/pi_tool.html (51).Corresponding ORF in SaPI11-bCorresponding ORFs were determined by relative position within the islands and/or similarity of the predicted gene products.Corresponding ORF in SaPIbovDescription (homolog)Expression detected in vitroExpression detected in vivo(amino acids)(kDa)sapi3_1/ear18520.21 (ear)selYYseb26631.4secEnterotoxinYYsapi3_318320.725(Terminase)Ysapi3_411313.436Ysapi3_517520.647Ysapi3_6728.258YYsapi3_719222.869sapi3_811313.410Ysapi3_921324.5711Ysapi3_109410.88sapi3_1112013.912Ysapi3_1247455.011(vapE)Ysapi3_1328933.313/1215(Replication protein)Ysapi3_1410612.716YYsapi3_15698.3YYsapi3_16556.0YYsapi3_179010.414(ORF 37 S. aureusphage φPVL)Ysapi3_18667.6(Cro-like repressor)YYsapi3_1911012.7(cI-like repressor)YYsapi3_2015218.1(ORF 153 of φSLT)YYsapi3_2114316.2(ABC transporter)YYseq24228.2seq′EnterotoxinYYsek24227.7sekEnterotoxinYYsapi3_int40747.6intintIntegraseYY1-a Determined using the Compute pI/Mw tool at ca.expasy.org/tools/pi_tool.html (51.Wilkins M.R. Gasteiger E. Bairoch A. Sanchez J.C. Williams K.L. Appel R.D. Hochstrasser D.F. Link A.J. 2-D Proteome Analysis Protocols. Humana Press, Totowa, NJ1998: 531-552Google Scholar).1-b Corresponding ORFs were determined by relative position within the islands and/or similarity of the predicted gene products. Open table in a new tab We have identified genes in SaPI3 according to the following nomenclature: pathogenicity island_ORF number (variant). Thus, the ninth ORF in SaPI3 is identified by the gene name sapi3_9. A mutant of this gene might be designated as sapi3_9(1). Sequential genes are identified using a hyphen (e.g. sapi3_10-15 is used to identify all SaPI3 ORFs from 10 through 15). Upon determination of the gene's function, the name will be altered to reflect that function, such as sapi3_int. (Exotoxins on the islands are identified according to their own nomenclature system.) If additional genes are identified on the island subsequent to its initial sequencing, those genes will be numbered sequential to those already identified, rather than renumbering all of the genes on the island. We have implemented this nomenclature system in our laboratory to promote systematic identification of SaPI genes, preclude confusion regarding identically numbered ORFs on different islands, and allow unambiguous assignation of expression data to SaPI genes. A comparison of SaPI3, SaPI1, and SaPIbov is shown in Fig. 1, and the corresponding ORFs are identified in Table I. The overall length of the three SaPIs is similar, although SaPIbov is larger on the 5′ end by 1915 bp. SaPIbov also has a different att site than SaPI1 and SaPI3. Two core regions of high (>92% identity) homology between all three islands were identified (Fig. 1). The first core region includes nucleotides 2974–6709 of SaPI1, 3113–6929 of SaPI3, and 5100–8745 of SaPIbov. Within this core region, SaPI1 has an additional 100 bp not present in SaPIbov (nucleotides 5909–6009 in SaPI1), whereas SaPI3 contains this 100-bp stretch as well as an additional 67 bp not present in either SaPI1 or SaPIbov (nucleotides 6063–6230). The second core region includes nucleotides 9380–10,284 of SaPI1, 9591–10,494 of SaPI3, and 11,419–12,429 of SaPIbov. All three islands contain int genes adjacent to the attR sites, and SaPI3 contains an enterotoxin gene (seb) in the same position as tstH in SaPI1 and SaPIbov. SaPI1 and SaPI3 appear to be even more closely related. In addition to the shared elements among all three islands and the identical attachment sites in SaPI1 and SaPI3, these two islands are highly homologous (>93% identity) at the right end (nucleotides 10,285–11,046 in SaPI1 and 10,494–11,254 in SaPI3; nucleotides 12,248–15,250 in SaPI1 and 12,891–15,953 in SaPI3) (Fig. 1). The ear(sapi1_1 and sapi3_1) and sek genes are also in the same relative positions to each other and to the att sites of SaPI1 and SaPI3. Although the function of ear is unknown, several properties of the gene suggest that it may have an important function in the life cycle of S. aureus. Its position and predicted product are conserved (∼75% identity at the amino acid level) among SaPI1, SaPI3, and SaPI4 (data not shown), it has the identical signal sequence as TSST-1, and it is secreted in abundant quantities by S. aureus RN4282. In addition to homology between SaPI1, SaPI3, and SaPIbov, the region encoding sapi3_3-9 and sapi3_15 shares extensive homology (>95% at the nucleotide level) to a matching region in SaPIn1/SaPIm1, whereas sapi3_10-14 are 87% or more similar to regions presumably of phage origin in strain Mu50. A locus of ∼900 bp adjacent to the attL site of SaPI3 was 95% identical to a phage 80α sequence adjacent to the putative phage amidase gene. However, this region of SaPI3 apparently does not encode for any protein. A ∼1.6-kb region SaPI3 with significant variance as compared with SaPI1 is found spanning the region between sapi3_17 and seq. Within this region is a stretch of ∼500 nucleotides that is nearly identical to a sequence from the recently identified φSLT, a temperate S. aureus phage encoding the Panton-Valentine leukocidin. Immediately upstream of int in both SaPI1 and SaPI3 is a 46-bp sequence conserved among staphylococcal phages φ11, φ13, φ42, and L54a (18.Carroll D. Kehoe M.A. Cavanagh D. Coleman D.C. Mol. Microbiol. 1995; 16: 877-893Crossref PubMed Scopus (60) Google Scholar, 19.Ye Z.H. Buranen S.L. Lee C.Y. J. Bacteriol. 1990; 172: 2568-2575Crossref PubMed Google Scholar, 20.Ye Z.H. Lee C.Y. J. Bacteriol. 1989; 171: 4146-4153Crossref PubMed Google Scholar). This sequence is the binding site for two phage φ11 proteins that regulate int expression, RinA and RinB (21.Ye Z.H. Lee C.Y. J. Bacteriol. 1993; 175: 1095-1102Crossref PubMed Google Scholar). In addition to the integrase and the noncoding regions with homology to phage DNA, several of the genes contained on SaPI3 suggest a mobile element of phage origin, perhaps a conglomeration of several phage elements (Table I). The terminase potentially encoded by sapi3_3 is homologous to the small subunit of identified terminases in the bacteriophages ρ15 (22.Chai S. Kruft V. Alonso J.C. Virology. 1994; 202: 930-939Crossref PubMed Scopus (29) Google Scholar) (52% similarity over 162 amino acids) and PBSX of Bacillus subtilis (23.McDonnell G. Wood H. Devine K. McConnell D. J. Bacteriol. 1994; 176: 5820-5830Crossref PubMed Google Scholar) (50% similarity over 102 amino acids). Sapi3_12 potentially encodes a product with high homology (54% similarity over 333 amino acids) to the virulence-associated protein (VapE) of Dichelobacter nodosus, a sheep pathogen (24.Katz M.E. Howarth P.M. Yong W.K. Riffkin G.G. Depiazzi L.J. Rood J.I. J. Gen. Microbiol. 1991; 137: 2117-2124Crossref PubMed Scopus (53) Google Scholar). (Sapi1_11 is also a VapE homolog 9.) This has led to the supposition that this gene was acquired by S. aureus through horizontal transfer from D. nodosus during co-colonization or infection of sheep (5.Novick R.P. Schlievert P. Ruzin A. Microbes Infect. 2001; 3: 585-594Crossref PubMed Scopus (115) Google Scholar) because the D. nodosus vap genes appear to reside on an integrated bacteriophage (25.Cheetham B.F. Katz M.E. Mol. Microbiol. 1995; 18: 201-208Crossref PubMed Scopus (198) Google Scholar, 26.Cheetham B.F. Tattersall D.B. Bloomfield G.A. Rood J.I. Katz M.E. Gene (Amst.). 1995; 162: 53-58Crossref PubMed Scopus (46) Google Scholar). The predicted product of sapi3_18 is 75% similar over 58 amino acids to a putative cro-like repressor of Streptococcus thermophilus bacteriophage Sfi21 (27.Bruttin A. Brussow H. Virology. 1996; 219: 96-104Crossref PubMed Scopus (42) Google Scholar, 28.Bruttin A. Desiere F. Lucchini S. Foley S. Brussow H. Virology. 1997; 233: 136-148Crossref PubMed Scopus (77) Google Scholar). Even stronger homology is seen between the predicted product of sapi3_19 and the cI-like repressor of S. the
Two Gram-negative bacteria with a high G+C content were isolated from soil in undergraduate microbiology classes by enriching for low nutrient growth and neonicotinoid pesticide tolerance. DNA from these isolates was purified and sequenced using a hybrid approach. Here we report the genome sequences of Pseudomonas alkylphenolica strain Neo and Variovorax sp. strain CSUSB.
Three Gram-negative bacteria and one Gram-positive bacterium were isolated from environmental samples in an undergraduate microbiology class on the basis of antibiotic resistance. Isolate DNA was purified, sequenced, and assembled using a hybrid approach. Here, we report the genomes of Acinetobacter johnsonii CSUSB1, Aeromonas hydrophila CSUSB2, Bacillus velezensis CSUSB3, and Comamonas thiooxydans CSUSB4.
Abstract Background Variovorax paradoxus is an aerobic soil bacterium frequently associated with important biodegradative processes in nature. Our group has cultivated a mucoid strain of Variovorax paradoxus for study as a model of bacterial development and response to environmental conditions. Colonies of this organism vary widely in appearance depending on agar plate type. Results Surface motility was observed on minimal defined agar plates with 0.5% agarose, similar in nature to swarming motility identified in Pseudomonas aeruginosa PAO1. We examined this motility under several culture conditions, including inhibition of flagellar motility using Congo Red. We demonstrated that the presence of a wetting agent, mineral, and nutrient content of the media altered the swarming phenotype. We also demonstrated that the wetting agent reduces the surface tension of the agar. We were able to directly observe the presence of the wetting agent in the presence and absence of Congo Red, and found that incubation in a humidified chamber inhibited the production of wetting agent, and also slowed the progression of the swarming colony. We observed that swarming was related to both carbon and nitrogen sources, as well as mineral salts base. The phosphate concentration of the mineral base was critical for growth and swarming on glucose, but not succinate. Swarming on other carbon sources was generally only observed using M9 salts mineral base. Rapid swarming was observed on malic acid, d-sorbitol, casamino acids, and succinate. Swarming at a lower but still detectable rate was observed on glucose and sucrose, with weak swarming on maltose. Nitrogen source tests using succinate as carbon source demonstrated two distinct forms of swarming, with very different macroscopic swarm characteristics. Rapid swarming was observed when ammonium ion was provided as nitrogen source, as well as when histidine, tryptophan, or glycine was provided. Slower swarming was observed with methionine, arginine, or tyrosine. Large effects of mineral content on swarming were seen with tyrosine and methionine as nitrogen sources. Biofilms form readily under various culture circumstances, and show wide variance in structure under different conditions. The amount of biofilm as measured by crystal violet retention was dependent on carbon source, but not nitrogen source. Filamentous growth in the biofilm depends on shear stress, and is enhanced by continuous input of nutrients in chemostat culture. Conclusion Our studies have established that the beta-proteobacterium Variovorax paradoxus displays a number of distinct physiologies when grown on surfaces, indicative of a complex response to several growth parameters. We have identified a number of factors that drive sessile and motile surface phenotypes. This work forms a basis for future studies using this genetically tractable soil bacterium to study the regulation of microbial development on surfaces.
ABSTRACT Staphylococcus aureus causes a wide variety of diseases. Major virulence factors of this organism include enterotoxins (SEs) that cause both food poisoning and toxic shock syndrome. Recently, a novel SE, tentatively designated SEL, was identified in a pathogenicity island from a bovine mastitis isolate. The toxin had a molecular weight of 26,000 and an isoelectric point of 8.5. Recombinant SEL shared many biological activities with SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered in subcutaneous miniosmotic pumps, but the protein lacked emetic activity. T cells bearing the T-cell receptor β chain variable regions 5.1, 5.2, 6.7, 16, and 22 were significantly stimulated by recombinant SEL.
Background Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylu. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode Acrobeloides maximus. Methodology We used a soil microcosm approach to expose A. maximus populations grown monoxenically on RFP labeled Escherichia coli in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using Caenorhabditis elegans N2. Recovered A. maximus populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome. Results A consistent core microbiome was extracted from A. maximus that differed from those in the bulk soil or the C. elegans associated set. Three genera, Ochrobactrum, Pedobacter, and Chitinophaga, were identified at high levels only in the A. maximus populations, which were less diverse than the assemblage associated with C. elegans. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence in situ hybridization (FISH) using probes specific for Ochrobactrum and Pedobacter stained bacterial cells in formaldehyde fixed nematode guts. Conclusions Three microorganisms were repeatedly observed in association with Acrobeloides maximus when recovered from soil microcosms. We isolated several Ochrobactrum sp. and Pedobacter sp., and demonstrated that they inhabit the nematode gut by FISH. Although their role in A. maximus is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria.
