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Acinetobacter baumanniiis a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. TheA. baumanniistrain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determinedA. baumanniiAbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of theA. baumanniiAbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology ofA. baumannii. [ABSTRACT FROM AUTHOR]

Actinobacillus actinomycetemcomitans causes periodontitis, a costly chronic infection that affects a large number of patients. The pathogenesis of this dental infection is a multifactorial process that results in a serious degenerative disease of the periodontium. Although significant progress has been achieved after the identification of this Gram-negative bacterium as the etiological agent of this infection, much remains to be done to understand in detail the bacterial factors and host-pathogen interactions involved in the pathogenesis of this disease. Classic research approaches have resulted in the identification of important virulence factors and cellular processes, although they have provided a rather narrow picture of some of the steps of this complex process. In contrast, a much wider picture could be obtained with the application of tools such as bioinformatics and genomics. These tools will provide global information regarding the differential expression of genes encoding factors and processes that lead to the pathogenesis of this disease. Furthermore, comparative genomics has the potential of helping us to understand the emergence and evolution of this human pathogen. This genome-wide approach should provide a more complete picture of the pathogenesis process of this disease, and will facilitate the development of efficient diagnostic, preventive, and therapeutic measures for this disease. [ABSTRACT FROM AUTHOR]

Acinetobacter calcoaceticus BD413 was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron-scavenging phenotype was associated with the secretion of iron-repressible catechol and the induction of a group of six outer membrane proteins with molecular weights ranging from 34 to 85 kDa. The amount of catechol produced was dependent on medium composition and iron stringency. The relation between iron limitation and lipase production was studied at the level of lipA transcription and extracellular lipase activity. In minimal medium, iron limitation slightly affected lipA expression but decreased exo-lipase activity significantly. However, if iron limitation and rich nitrogen sources were simultaneously present in the culture media, the production of lipase was increased approximately 4 times.

The bacterial enzyme histidine decarboxylase (Hdc) catalyses the conversion of histidine into histamine. This amine is essential for the biosynthesis of iron chelators (siderophores) and is an important cause of food poisoning after consumption of fish contaminated with histamine-producing bacteria. In this work we compared different methods for detecting histamine secreted by different bacterial strains. The presence of histamine in the culture supernatant of Vibrio anguillarum, which produces Hdc and secretes the histamine-containing siderophore anguibactin, was detected by thin-layer chromatography. Similar results were obtained using the culture supernatant of the Acinetobacter baumannii 19606 prototype strain that secretes the histamine-containing siderophore acinetobactin. Conversely, histamine was not detected in the culture supernatant of an isogenic V. anguillarum Hdc mutant and the A. baumannii 8399 strain that secretes a catechol siderophore different from anguibactin and acinetobactin. These results were confirmed by capillary gas chromatography/mass spectrometry. However, all these strains tested positive for histamine secretion when cultured on differential plating media containing histidine and a pH indicator, which were specifically designed for the detection of histamine-producing bacteria. The pH increase of the medium surrounding the bacterial colonies was however drastically reduced when the histidine-containing medium was supplemented with peptone, beef extract, and glucose. The histidine-containing culture supernatants of the A. baumannii and V. anguillarum strains showed an increase of about two units of pH, turned purple upon the addition of cresol red, and contained high amounts of ammonia. Escherichia coli strains, which are Hdc negative and do not use histidine as a carbon, nitrogen, and energy source, gave negative results with the differential solid medium and produced only moderate amounts of ammonia when cultured in the presence of excess histidine. This study demonstrates that, although more laborious and requiring some expensive equipment, thin-layer and gas chromatography/mass spectrometry are more accurate than differential media for detecting bacterial histamine secretion. The results obtained with these analytical methods are not affected by byproducts such as ammonia, which are generated during the degradation of histidine and produce false positive results with the differential plating media.

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication., (Copyright 1999 Academic Press.)

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

Haemophilus influenzae biogroup aegyptius (H. aegyptius) is the etiological agent of Brazilian purpuric fever (BPF), a recently described pediatric disease that is often fatal. The vascular destruction that occurs in this disease is a distinctive trait, and little is known about the mechanism(s) of the overwhelming purpura fulminans that causes the high mortality associated with this pediatric infection. Iron is an essential micronutrient for nearly all living cells, and the mechanisms used by bacteria to acquire and internalize iron are often associated with virulence. Therefore, the focus of our studies is the molecular characterization of the iron uptake system used by H. aegyptius. Specifically, we are investigating the high-affinity transferrin binding proteins in the bacterial outer membrane, components of ABC transporter systems, and a possible regulatory mechanism for the genes encoding these proteins. A detailed understanding of the molecular nature of the regulatory genetic components and proteins involved in the acquisition of iron will broaden the knowledge of the pathogenesis of the disease caused by H. aegyptius and will also lead to a better understanding of the nature of other infections that affect the vascular system.

Actinobacillus actinomycetemcomitans was found to express a polypeptide immunologically related to the Neisseria gonorrhoeae FbpA iron binding protein. In addition, the expression of hitB and hitC homologs was detected by Northern blot analysis. This periodontal pathogen also expresses a polypeptide homologous to the 31-kDa Haemophilus influenzae protein, which shows amino acid sequence homology with the FimA and YfeA proteins from Streptococcus parasanguis and Yersinia pestis, respectively. Both A. actinomycetemcomitans protein homologs were located within the periplasmic space, and their synthesis was regulated by the iron and hemin concentration of the culture medium. Southern and Western blot analysis together with molecular cloning revealed the presence of a Fur-like repressor, which may control the iron regulation of gene expression in this bacterium. Cultivation in the presence of hemin or Congo red revealed the ability of this organism to bind hemin. This binding activity was further confirmed by isolating Escherichia coli DH5 alpha clones that produced red and brown colonies on agar plates containing Congo red and hemin, respectively, after transformation with an A. actinomycetemcomitans gene library.

Histamine production in bacteria-contaminated fish is the result of the presence of bacterial histidine decarboxylase activity, which converts histidine present in muscle proteins to histamine. The fish pathogen Vibrio anguillarum harbors a plasmid-encoded histidine decarboxylase gene (angH) that is essential for biosynthesis of the siderophore anguibactin. However, the role of angH in histamine biosynthesis by this pathogen has not been fully determined. Thus, the objectives of this study were to monitor the production and release of histamine by the wild-type as well as by a plasmidless strain and angH isogenic mutants generated by allelic exchange. Reverse transcription polymerase chain reaction showed that only the wild-type strain expressed angH, while no angH message was detected in the mutants and the plasmidless derivative. The iron uptake-deficient phenotype of one of the angH mutants confirmed the location of the mutation and the unique role of this gene in iron acquisition. Thin-layer chromatography, gas chromatography, and mass spectrometry showed that histamine was released by the strain harboring a wild-type angH gene when grown in excess histidine. This biogenic amine was not detected in the culture supernatants of the plasmidless derivative and the angH mutant when cultured under the same experimental conditions. These results indicate that angH is essential for histamine biosynthesis in V. anguillarum, a compound responsible for food poisoning and potentially involved in bacterial virulence. Thin-layer chromatography of wild-type culture supernatants and beta-galactosidase assays using the isogenic angH mutant demonstrated that the expression of this gene is independent of the histidine concentration of the medium under both iron-rich and iron-limiting conditions., (Copyright 1998 Academic Press.)

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJM1 which encodes a histidine decarboxylase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44,259.69 Da. The amino acid sequence has extensive homology with the pyridoxal-P-dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3-HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJM1 iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A. V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron-limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).

Synthesis of the 86-kDa FatA outer membrane protein is repressed under iron-rich conditions. Complementation of transposition mutants derived from clones containing the pJM1 iron uptake region revealed the existence of an antisense RNA, RNA alpha. This RNA is only expressed under iron-rich conditions and acts as a negative regulator of FatA synthesis, with slight but discernible decrease in the steady-state level of fatA mRNA determined by RNase protection and by Northern blot analysis. Primer extension experiments revealed that the level of several possible fatA transcripts was reduced in the presence of RNA alpha. In addition, we found that fatA mRNA expression is slightly reduced in the presence of Escherichia coli Fur. We have identified and cloned a chromosomally encoded fur-like gene in Vibrio anguillarum.

Complementation of insertion mutants showed that the polypeptides FatD, FatC, FatB, and FatA are essential for the iron-transport process encoded by pJM1. Sequence analysis followed by homology studies indicated that transport of ferric anguibactin into Vibrio anguillarum 775 follows the same mechanism as reported for transport of Fe(3+)-hydroxamates, Fe(3+)-catecholates, ferric dicitrate, and vitamin B12 into Escherichia coli. Homology of FatA, part of the receptor complex, to seven E. coli receptor proteins involved in uptake of siderophores and vitamin B12 supports the idea of a common ancestral gene. A "TonB-Box" was found in FatA suggesting the existence of a TonB-like protein function in V. anguillarum. A high homology in the primary structure of FatB to FhuD, FecB, FepB, and BtuE suggests that FatB is the anguibactin-binding protein located in the periplasmic space. FatD and FatC are polytopic integral membrane proteins. According to their homologies to other proteins from other transport systems, they may be involved in the translocation of ferric anguibactin across the cytoplasmic membrane.

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.