Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish.

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri.

AIMS: To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons. METHODS AND RESULTS: The Edw. ictaluri rrn operons were identified from a 5-7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I-CeuI enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri. CONCLUSIONS: The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family. SIGNIFICANCE AND IMPACT OF THE STUDY: This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.

Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force.

We have characterized the interaction of the Neisseria meningitidis TonB-dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [125I]-Hb revealed that the bipartite receptor has a single binding site for Hb (Kd 150 nM). Competitive binding assays using heterologous Hbs revealed that HpuAB Hb recognition was not species specific. The binding kinetics of Hb to HpuAB were dramatically altered in a TonB- mutant and in wild-type meningococci treated with the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that TonB and an intact proton motive force are required for normal Hb binding and release from HpuAB. Our results support a model in which both HpuA and HpuB are required to form a receptor complex in the outer membrane with a single binding site, whose structure and ligand interactions are significantly affected by the TonB-mediated energy state of the receptor.

Reduced virulence of a Bordetella bronchiseptica siderophore mutant in neonatal swine.

One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.

Phase variation of HpuAB and HmbR, two distinct haemoglobin receptors of Neisseria meningitidis DNM2.

We have previously described HpuAB, a two-component receptor that mediates binding to haemoglobin (Hb), haemoglobin-haptoglobin (Hb-Hp) and apo-haptoglobin (Hp). In this communication, we constructed non-polar mutations in the hpuA and hpuB loci to examine the individual roles of HpuA and HpuB. Our results indicate that both HpuA and HpuB are required for the acquisition of Fe from Hb and Hb-Hp. We isolated Hb utilization-positive (Hb+) variants of our Hb utilization-negative (Hb-) hpu mutants at a frequency of 10(-3) and demonstrated that the Hb+ phenotype resulted from the expression of a second Hb receptor, HmbR. Expression of HmbR in DNM2 was found to be controlled by translational frameshifting involving a polyguanine (G) tract located within the hmbR locus. The hpuA locus also contains a poly(G) tract, which suggested that meningococci could phase vary each Hb receptor independently by slip-strand mispairing in the poly(G) tracts found in hpuA and hmbR. Thus, we isolated a naturally occurring Hb- variant of DNM2, designated DNM2 Hb-, which did not express either HpuAB or HmbR. Hb+ variants of DNM2Hb- were selected and examined for HpuAB and HmbR expression. In each instance, acquisition of HpuAB or HmbR expression was correlated with phase variation in the poly(G) tract of each Hb receptor.

Transport of intact porphyrin by HpuAB, the hemoglobin-haptoglobin utilization system of Neisseria meningitidis.

The meningococcal hemA gene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of the hemA mutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA- HpuAB- double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.

Identification and molecular analysis of lbpBA, which encodes the two-component meningococcal lactoferrin receptor.

We identified lbpB, encoding the lipoprotein component of the meningococcal lactoferrin receptor. An LbpB mutant was unable to acquire Fe from lactoferrin and exhibits decreased surface binding to lactoferrin. Primer extension and reverse transcription-PCR analysis indicate that lbpB and lbpA are cotranscribed on a polycistronic Fe-repressible mRNA.

Analysis of the alcABC operon encoding alcaligin biosynthesis enzymes in Bordetella bronchiseptica.

We previously cloned a B. bronchiseptica (Bb) genomic DNA fragment that complements a Bb alcaligin biosynthesis mutant, and reported the identification of a gene, alcA, with predicted protein sequence similarity to siderophore biosynthesis enzymes from other organisms. In the present study we show that further nt sequencing of this region revealed two open reading frames (ORFs) 3' to alcA that encode putative proteins AlcB and AlcC, with significant sequence similarity to the aerobactin biosynthesis enzymes IucB and IucC, respectively. RT-PCR analysis indicated that the three ORFs are encoded on a single transcript, and that this operon is repressed at the transcriptional level by Fe. Primer extension analysis placed the transcriptional start point (tsp) 35 nt from the 5' end of the Fur consensus sequence and 188 nt from the putative start of translation of AlcA.

Molecular characterization of hpuAB, the haemoglobin-haptoglobin-utilization operon of Neisseria meningitidis.

We previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5' to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5' to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5' to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA.

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.

Cloning and characterization of the Actinobacillus actinomycetemcomitans gene encoding a heat-shock protein 90 homologue.

In a search for clones from a lambda gt11 expression library of Actinobacillus actinomycetemcomitans (Aa) genomic DNA that expressed epitopes from a 70-kDa iron-repressible membrane protein, we inadvertently identified clones that encoded a member of the 90-kDa heat-shock protein (HSP 90) family. The gene appears to encode a homologue of HtpG, as the nucleotide sequence has approximately 70% identity with the Escherichia coli (Ec) and Vibrio fischeri htpG. Growth of an Aa htpG insertion mutant at 42 degrees C was reduced to 50% of the parent strain, similar to an Ec htpG deletion mutant. These data suggest that Aa HtpG performs a function similar to Ec HtpG.

Effect of protoporphyrin IX limitation on porphyromonas gingivalis.

Porphyromonas gingivalis has been shown to require hemin or hemoglobin for in vitro growth. We have previously shown that protoporphyrin IX and inorganic iron can replace the hemin requirement, suggesting that the hemin requirement of this microorganism is actually a porphyrin requirement. We examined the effect of protoporphyrin IX limitation to P. gingivalis strain A7A1-28 in the presence of sufficient iron on growth characteristics, proteolytic enzyme production, virulence in a mouse abscess model, and expression of membrane proteins. Bacterial cells were grown in medium varying between 0 to 5 microM reduced growth by at least 50%. Protoporphyrin IX availability did not affect proteolytic enzyme production or virulence in a mouse abscess model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane preparations demonstrated that protoporphyrin IX limitation induced the expression of new proteins at 42, 34, 30, 29, and 18 kDa and suppressed the production of proteins at 47, 27, 17, and 15 kDa. These studies suggest that in vivo protoporphyrin availability may modulate membrane protein expression and in turn affect host immune responses against P. gingivalis.

Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production.

The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced. The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846. Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron. The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin.

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.

Nonradioactive colony lift-hybridization assay for detection of Bordetella bronchiseptica infection in swine.

Current methods for the isolation and identification of Bordetella bronchiseptica from clinical samples are time-consuming and are based, in part, on subjective observations. We describe the use of a Bordetella-specific DNA probe in a nonradioactive colony lift-hybridization assay for the identification of B. bronchiseptica. Eleven of 82 clinical specimens were found to contain B. bronchiseptica by this method, while only 5 of these were reported to contain the organism when the specimens were analyzed by traditional methods. The chromosomal fragment containing a sequence complementary to the probe appeared to be conserved in B. bronchiseptica isolates from swine from a variety of sources. The assay is more rapid than culture and biochemical testing since it can be performed directly on primary culture plates, even when they are heavily contaminated with other bacterial species. Only minimal training is required to accomplish the assay successfully, and the results are easy to interpret.

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).

Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.

Growth of Moraxella catarrhalis with human transferrin and lactoferrin: expression of iron-repressible proteins without siderophore production.

Moraxella (Branhamella) catarrhalis, a mucosal pathogen closely related to Neisseria species, is a prominent cause of otitis media in young children and lower respiratory tract infections in adults. In this study, we investigated whether M. catarrhalis can compete for iron bound to human transferrin or human lactoferrin in a manner similar to that utilized by Neisseria meningitidis and Neisseria gonorrhoeae. Our studies demonstrated that M. catarrhalis obtains iron from these serum carrier proteins and also maintains growth with ferric nitrate in vitro. Furthermore, we report that when M. catarrhalis is grown under iron-limited conditions, the bacteria express new outer membrane proteins that are not detected in membranes of organisms cultured in an iron-rich environment. We have shown that these are iron-repressible proteins since they are not induced by other environmental stresses and the expression of these proteins is repressed when a source of iron is provided for iron-limited bacteria. The iron-repressible proteins are expressed in the absence of any detectable siderophore production. These iron-repressible proteins may be important for the acquisition and utilization of iron in vivo, which could allow M. catarrhalis to colonize and survive on human mucosal surfaces.

Insertional inactivation of the gene for the meningococcal lactoferrin binding protein.

The lactoferrin binding protein (LBP) of Neisseria meningitidis (the putative meningococcal receptor for human lactoferrin, LF), has been previously characterized as an outer-membrane protein of approximately 105 kDa. Using N-terminal amino acid sequence to generate an oligonucleotide probe, a clone from a lambda gt11 phage library was isolated. This clone was subjected to shuttle mutagenesis, in which an erythromycin mini-transposon was used to interrupt the LBP coding sequence. This insertion mutation was introduced into the meningococcus. A N. meningitidis strain that carried this transposon insertion no longer produced the 105 kDa protein. The absence of this protein was correlated with the inability to bind LF or to use LF as an iron source. The LBP mutant was able to grow with other Fe sources and demonstrated no other visible membrane protein alterations. These data confirm the suggestion that LBP is the meningococcal receptor.