Search

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

Porphyromonas gingivalis is a major pathogen of periodontal diseases, including periodontitis. We have investigated the effect of P. gingivalis infection on the PI3K/Akt (protein kinase B) signaling pathway in gingival epithelial cells. Here, we found that live P. gingivalis, but not heat-killed P. gingivalis, reduced Akt phosphorylation at both Thr-308 and Ser-473, which implies a decrease in Akt activity. Actually, PI3K, which is upstream of Akt, was also inactivated by P. gingivalis. Furthermore, glycogen synthase kinase 3α/β, mammalian target of rapamycin, and Bad, which are downstream proteins in the PI3K/Akt cascade, were also dephosphorylated, a phenomenon consistent with Akt inactivation by P. gingivalis. However, these events did not require direct interaction between bacteria and host cells and were independent of P. gingivalis invasion into the cells. The use of gingipain-specific inhibitors and a gingipain-deficient P. gingivalis mutant KDP136 revealed that the gingipains and their protease activities were essential for the inactivation of PI3K and Akt. The associations between the PI3K regulatory subunit p85α and membrane proteins were disrupted by wild-type P. gingivalis. Moreover, PDK1 translocation to the plasma membrane was reduced by wild-type P. gingivalis, but not KDP136, indicating little production of phosphatidylinositol 3,4,5-triphosphate by PI3K. Therefore, it is likely that PI3K failed to transmit homeostatic extracellular stimuli to intracellular signaling pathways by gingipains. Taken together, our findings indicate that P. gingivalis attenuates the PI3K/Akt signaling pathway via the proteolytic effects of gingipains, resulting in the dysregulation of PI3K/Akt-dependent cellular functions and the destruction of epithelial barriers.

Silkworm larva has recently been recognized as an alternative model animal for higher mammals to evaluate the effects of antibiotics. In this study, we examined the efficacy of the bacteriophage (phage) therapy, which harnesses phages as antibacterial agents, against Staphylococcus aureus infections, using the silkworm larval infection model. Two newly isolated staphylococcal phages, S25-3 and S13', were used as therapeutic phage candidates. They were assigned to two different lytic phage genera, Twort-like and AHJD-like viruses, based on their morphologies and the N-terminal amino acid sequences of the major capsid proteins. Both had a broad host range and strong lytic activity and showed preservative quality. Administration of these phages alone caused no adverse effects in the silkworm larvae. Moreover, the viruses showed life-prolonging effects in the silkworm larval infection model 10 min, 6 h, 12 h, and 24 h following infection. Such phage effects in the silkworm larval model were almost paralleled to the therapeutic efficacies in mouse models. These results suggest that phages S25-3 and S13' are eligible as therapeutic candidates and that the silkworm larval model is valid for the evaluation of phage therapy as well as mouse models.

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.

Actinobacillus actinomycetemcomitans is a gram-negative pathogen that causes human severe periodontitis. The most intensively studied virulence factor of this bacterium is a pore-forming cytotoxin termed leukotoxin. Production of leukotoxin varies considerably among A. actinomycetemcomitans strains. Interstrain variations in leukotoxin production are thought to derive from differences in structure of the leukotoxin promoter region. In addition, several studies showed that culture conditions are also important factors influencing leukotoxin production levels. Export of A. actinomycetemcomitans leukotoxin across the cell envelope is likely mediated by a type I secretion system. Furthermore, it has also been suggested that outer membrane vesicles are involved in extracellular localization of the leukotoxin. This review summarizes current knowledge on the genetic and environmental factors affecting leukotoxin production and the export pathway for extracellular localization of leukotoxin.

Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria–Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS–PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.Key words: chromosome fragmentation, DNA excretion, defective prophage, PBSX, horizontal gene transfer.

Background Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. Methods To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. Results Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p

We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-Bacteroides/P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.

Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells)−1 to 3·1 pmol (mg dry wt cells)−1, which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.

Actinobacillus actinomycetemcomitans, a member of the gamma subclass of the Proteobacteria, has been implicated as the agent responsible for human periodontitis. In this study, A. actinomycetemcomitans 301-b was grown in fructose-limited chemostat cultures under anaerobic [redox potential (E(h))-400 mV] and microaerobic (E(h)= -200 mV) conditions to characterize its energy metabolism. Effects of K(+) and Na(+) on growth and metabolism were also examined. In a control medium containing 5.2 mM K(+) and 24 mM Na(+), the molar growth yield on fructose (Y(fructose)) of microaerobic cultures was 1.3 times higher than the yield of anaerobic cultures at Dor =0.10 h(-1), but the difference in the Y(fructose) between microaerobic and anaerobic cultures decreased at Dor =0.10 h(-1). When the ATP yield from fermentation was estimated from the amounts of fructose consumed and acetate formed, the value of the microaerobic culture (2.49 mol ATP produced per mol fructose consumed) was lower than the anaerobic value [3.13 mol ATP (mol fructose)(-1)]. Therefore, ATP production from fermentation could not account for the increase in the Y(fructose) at D0.10 h(-1) and thus additional ATP was expected to be generated via respiration. Assuming that the Y(ATP) (g cells formed per mol ATP synthesized) was similar between anaerobic and microaerobic cultures, the estimated ATP yield from respiration was between 1.2 and 2.0 mol ATP (mol fructose)(-1) below D=0.10 h(-1) and decreased to 0.3 mol ATP (mol fructose)(-1) when D was increased to 0.19 h(-1). Such growth-rate-dependent decreases in the Y(fructose) and the estimated ATP production from respiration were also observed in a high-Na(+) (5.2 mM K(+) and 106 mM Na(+)) culture but not in a high-K(+) (81 mM K(+) and 24 mM Na(+)) culture. In the high-K(+) culture, the microaerobic Y(fructose) was 1.4-2.0 times higher than the anaerobic value and the respiration-derived ATP yield was estimated to be between 1.2 and 1.9 mol ATP (mol fructose)(-1) over a wide range of dilution rate. These results suggest that higher concentrations of extracellular K(+) are required for the respiration to occur in rapidly growing cells of A. actinomycetemcomitans.

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus . Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constrants estimated for TMR-labeled GTF at pH 6.8 and 25°C in the absence and presence of sucrose were 9.2 and 13.3 s −1 , respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (≥25 s −1 ) under comparable conditions.

Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis , which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis , hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production.

歯学領域では, Actinobacillus actinomycetemcomitans が, 歯周病を惹起する細菌の一つとして注目されている。本稿では, この細菌の主要な病原因子の一つであるロイコトキシンと歯周組織への定着に関与する線毛について概説した。ロイコトキシンはヒトの単球と多形核白血球を特異的に傷害する蛋白毒素であり, 大腸菌のα-ヘモリジンに代表されるRTX毒素ファミリーに属する。ロイコトキシンの発現は, 培養条件の違いによって大きく変動する。ケモスタットを用いて増殖環境を制御した実験では, ロイコトキシン産生は増殖速度に依存し, 重炭酸イオンで促進され, 嫌気ないし微好気条件で至適となり, さらに, カタボライト抑制様の機構で調節される結果が得られている。このような複雑な毒素発現調節は他のRTX毒素と比べても特異である。一方, A. actinomycetemcomitans の線毛についての情報は極めて限られていた。それは, 線毛の発現が新鮮分離株でしかみられず, 継代培養によって消失するからである。最近になって, ようやく, 線毛の精製と遺伝子クローニングが成功した。その遺伝子解析の結果からは, いくつかのグラム陰性菌でみられるタイプIV線毛との共通性がみえてきた。

KVP40 is a broad-host-range vibriophage forming plaques on strains of at least eight Vibrio and one Photobacterium species. A spontaneous KVP40-resistant mutant, R4000, derived from Vibrio parahaemolyticus 1010 lacked a 26-kDa outer membrane protein designated OmpK. KVP40 was inactivated by outer membrane and OmpK prepared from 1010, but not by outer membrane from R4000. These results strongly suggest that OmpK is the receptor for KVP40. Immunoblotting analyses using an anti-OmpK rabbit serum revealed that OmpK or its homologs of molecular masses 25-29 kDa were distributed widely among Vibrio and Photobacterium strains including those naturally resistant to KVP40.

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.

Actinobacillus actinomycetemcomitans strain 310-TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae-deficient mutant 310-DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310-TR formed tight and spherical microcolonies, while 310-DF produced looser ones. These findings suggest that fimbriae are not essential for the surface-adherent growth but are required for enhancing cell-to-surface and cell-to-cell interactions to stabilize the biofilm. Treatment of the 310-DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.

A remarkable cell shape change was observed inBacillus subtilisstrain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0·1 % KNO3and the shape-changed cells grown without KNO3revealed that the expression of the branched-chainα-keto acid dehydrogenase complex (coded by thebfmBgene locus) was elevated in the shape-changed cells. Inactivation of thebfmBlocus resulted in the repression of cell shape change, and cells in whichbfmBexpression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0·1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.

A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA-DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4-type phages having a more elongated head.

Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

Cell surface-associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris-buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N-terminal sequencing to be the 34 and 39 kDa outer membrane proteins, the GroEL-like protein, and a 47 kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid-exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low-molecular-weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54-kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304-a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N-terminal part of 26 amino acids is leader peptide. The N-terminal sequence of mature Flp exhibited some similarity to type-IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type-IV prepilin, and a gene encoding a protein homologous to type-IV prepilin-like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.

The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced. The gene consisted of 789 nucleotides encoding 263 amino acids. Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da. Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.

A broad-host-range vibriophage KVP40 originally isolated on Vibrio parahaemolyticus 1010 was restricted and modified by strains of at least five Vibrio and one Photobacterium species. 1010 was a non-restricting host. An anti-restriction mutant KVP40 aar1 was isolated after propagating the phage on a restricting host, V. anguillarum VIB36. KVP40 aar1 grown on either 1010 or VIB36, as well as the parental phage grown on VIB36, showed much higher efficiencies of plating on all the restricting hosts as compared with the parental phage grown on 1010, indicating that these restricting hosts probably share a common restriction-modification system active in vivo on KVP40.