Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.

Iron is an essential element for nearly all living cells. Thus, the ability of bacteria to utilize iron is a crucial survival mechanism independent of the ecological niche in which the microorganism lives, because iron is scarce both in potential biological hosts, where it is bound by high-affinity iron-binding proteins, and in the environment, where it is present as part of insoluble complex hydroxides. Therefore, pathogens attempting to establish an infection and environmental microorganisms must all be able to utilize the otherwise unavailable iron. One of the strategies to perform this task is the possession of siderophore-mediated iron uptake systems that are capable of scavenging the hoarded iron. This metal is, however, a double-edged sword for the cell because it can catalyze the production of deadly free hydroxyl radicals, which are harmful to the cells. It is therefore imperative for the cell to control the concentration of iron at levels that permit key metabolic steps to occur without becoming a messenger of cell death. Early work identified a repressor, Fur, which as a complex with iron repressed the expression of most iron uptake systems as well as other iron-regulated genes when the iron concentration reached a certain level. However, later work demonstrated that this regulation by Fur was not the only answer under low-iron conditions, there was a need for activation of iron uptake genes as well as siderophore biosynthetic genes. Furthermore, it was also realized that in some instances the actual ferric iron-siderophore complex induced the transcription of the cognate receptor and transport genes. It became evident that control of the expression of iron-regulated genes was more complex than originally envisioned. In this review, I analyze the processes of signal transduction, transcriptional control, and posttranscriptional control of iron-regulated genes as reported for the ferric dicitrate system in Escherichia coli; the pyochelin, pyoverdin, and enterobactin systems in Pseudomonas species; the irgB system in Vibrio cholerae; and the plasmid-mediated anguibactin system in Vibrio anguillarum. I hope that by using these diverse paradigms, I will be able to convey a unifying picture of these mechanism and their importance in the maintenance and prosperity of bacteria within their ecological niches.

Regulation of angR, a gene with regulatory and biosynthetic functions in the pJM1 plasmid-mediated iron uptake system of Vibrio anguillarum.

AngR and the product(s) encoded in the trans-acting factor (TAF) region are necessary for the full expression of the pJM1 plasmid-mediated anguibactin iron-uptake system in Vibrio anguillarum (Va). In this report, we analyzed the factors that affect the expression of the angR gene. Northern blot analysis showed that angR encodes a 3.1-kb transcript which is expressed only under iron-limiting conditions. Measurement of steady-state RNA levels show that, under iron-limiting conditions, angR is positively regulated at the transcriptional level by product(s) of the Va TAF region. However, this enhancement of angR expression by TAF does not occur at high levels of the AngR protein, as assessed by using an angR::lacZ fusion in the presence of a construct containing angR under the control of ptac. We also report that repression of angR by iron could possibly be mediated by an endogenous Va antisense RNA beta, which contains a stem-loop structure complementary to the stem-loop structure located at the 5' end of angR.

Transcription of the region encoding the ferric dicitrate-transport system in Escherichia coli: similarity between promoters for fecA and for extracytoplasmic function sigma factors.

We have analyzed the molecular mechanism of regulation of the ferric dicitrate transport system in Escherichia coli (Ec), by studying the transcription of the regulatory and structural genes under various environmental conditions, and by determining the location of their transcriptional start points and promoter regions. We report here that the main species observed in Northern hybridization analyses were a 2.5-kb mRNA, encoded by the outer membrane protein receptor gene fecA, and a 1.5-kb mRNA encoded by a region including the fecIR genes. The synthesis of the 2.5-kb fecA mRNA is regulated by both citrate and iron. Furthermore, transcription of fecA is dependent on the presence of FecI. The promoter region for the fecA mRNA, a likely site of action for FecI, is not related to the consensus promoter region for sigma 70 RNA polymerase in Ec K-12. However, it shows greatest similarity with promoters of genes regulated by a new sub-family of sigma factors, i.e., the extracytoplasmic function (ECF) sigma factors, which are associated with the expression of genes involved in extracytoplasmic functions, suggesting that FecI may act as a specialized sigma factor. We also show that the fecB,C,D,E transport genes are linked in operon fashion to fecA. Since the levels of the fecB,C,D,E RNAs are extremely low, as compared to the level of fecA mRNA, it is likely that processing from the 3' end must occur and stop near the end of fecA where a hairpin structure is located.

Characterization of the recA gene of Vibrio anguillarum.

We have cloned and sequenced the recA gene from two strains, 775 and 531A, of the fish pathogen, Vibrio anguillarum. Although both strains showed different sensitivities to methyl methanesulfonate (MMS), the recA genes were identical. In vitro expression of the V. anguillarum recA gene produced a polypeptide of about 40 kDa, in agreement with the value obtained from the nucleotide sequence. We identified the transcription start point by primer extension. The promoter for the recA gene mapped to an SOS regulatory element. The presence of an SOS box suggests that a LexA-like mediated response system may exist in V. anguillarum. The deduced RecA amino acid sequence is highly homologous with Escherichia coli RecA and other RecA proteins. Domains important in RecA function are conserved. We provide a comparative analysis of the activities and features of RecA analogs from a variety of species. We observed that certain residues that could be important in protein conformation are conserved in RecA proteins across a diverse range of bacterial species.

Regulation of the expression of bacterial iron transport genes: possible role of an antisense RNA as a repressor.

Expression of the iron transport gene, fatB and fatA, in the bacterium Vibrio anguillarum 775 is negatively regulated by the iron concentration in the medium. Here, we show that iron represses fatB and fatA mRNA levels and concomitantly induces the synthesis of an antisense RNA (RNA alpha). The presence of RNA alpha correlates with the inhibition of FatA protein synthesis and thus may play a role in the iron repression of fatA expression. Since the 5' end of RNA alpha maps 125 nucleotides upstream from the start codon of fatA and this RNA also extends into the coding region of fatB, it may also be involved in the iron regulation of fatB expression. RNA alpha may thus constitute a novel component of the bacterial iron regulatory circuit.

Molecular cloning of the replication terminus of the plasmid R6K.

Analyses of the intermediates of DNA replication of the R6K plasmid derivatives, RSF1040, RJHC12 and RJHC26 demonstrate the transient accumulation of open circular DNA molecules with a discontinuity in either the plus or the minus strand of the DNA. The location of this discontinuity is nonrandom and is near the terminus of DNA replication. The discontinuity is not due to the activation of a relaxation complex since neither RJHC12 nor RJHC26 are relaxable. The replication terminus is functional in a clone containing approx. a 2000 bp sub-fragment of the HindIII-2 fragment of R6K. The replication terminus temporarily arrests the progression of replication fork of the unidirectionally replicating plasmid pBR313 at a region approx. 800 bp from HindIII site located nearest to the BamHI site of the vector. Subcloning experiments reveal that the upper limit of the replication termination sequence is 216 bp in length.

A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron.

The angR locus in Vibrio anguillarum encodes a trans-acting transcriptional activator which modulates several Fe2(+)-regulated loci in the anguibactin biosynthesis gene cluster. In this paper, the complete nucleotide (nt) sequence of the angR gene and deduced amino acid (aa) sequence of the AngR protein are presented. A region upstream from the angR gene is shown to have similarity with Fe2(+)-regulated operators in Escherichia coli which bind the Fur protein. The involvement of a Fur-like regulator is supported by transcription analysis which show that angR itself is Fe2(+)-regulated. The aa sequence of the AngR protein predicts a helix-turn-helix motif which shows striking homology with prokaryotic DNA-binding proteins, particularly the lambda and P22 Cro proteins. In addition, there are two 18-nt regions, upstream from the angR gene, which show similarity with the OR1 and OR2 operators of P22 cro. These regions overlap with, respectively, the -35, -10 region and the putative Fur-binding region upstream from angR. These results suggest that AngR may be a DNA-binding protein which modulates Fe2(+)-regulated transcription and is itself Fe2(+)-regulated at the transcriptional level.

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds.

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.

Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags.

PURPOSE: Acinetobacter calcoaceticus subspecies anitratus (A. anitratus) can cause nosocomially and community acquired pneumonia. Source identification of the organism is often difficult. An outbreak of respiratory infection and colonization with A. anitratus affecting 93 ventilated patients in all six of a hospital's intensive care units (ICUs) over 10 months is described. PATIENTS AND METHODS: In April 1984, the infection control staff started to review positive culture results from all patients in all ICUs. At this point, information on significant isolates was recorded by patient, site, date, genus and species, and antimicrobial susceptibility. During the month of August 1984, an increased number of A. anitratus isolates from sputum began to be detected. Information was expanded to include the date of hospital admission, ICU admission, intubation, and extubation; the dates and types of all surgical procedures; the results and dates of all prior sputum cultures; and the use of nebulized bronchodilator medications. Monthly numbers of cases were compared for four months prior to the outbreak, during the outbreak, and for seven months after the outbreak. Plasmid DNA from isolates was prepared, electrophoresed, and visualized. Isolates were designated according to the molecular weights of visualized plasmids. RESULTS: Barrier precautions and improved staff handwashing did not diminish the frequency of new cases. When pasteurized, reusable ventilator circuits and resuscitation bags were cultured for the possibility of low-level contamination, 18 percent were positive for A. anitratus. Terminal ethylene oxide sterilization of these devices was associated with prompt control of the outbreak. Plasmid DNA analysis of isolates from patients involved in the outbreak, contaminated devices, and the hands of personnel responsible for device disinfection revealed two predominant plasmid profiles. After outbreak control, isolates with these profiles were found much less frequently in patient specimens. CONCLUSION: Contaminated, reusable ventilator support equipment may be a leading cause for the extent of A. anitratus in the sputum of intubated patients. This problem is potentially correctable by the use of terminal etyhlene oxide sterilization of reusable ventilator circuits and resuscitation bags.

Plasmid DNA analysis of Staphylococcus epidermidis isolated from blood and colonization cultures in very low birth weight neonates.

We prospectively studied the course of colonization and sepsis with Staphylococcus epidermidis among 29 very low birth weight neonates undergoing prolonged umbilical catheterization. S. epidermidis bacteremia occurred in 7 patients. In 6 bacteremia was preceded by positive colonization cultures. Isolates obtained from nares, base of umbilicus, umbilical catheter entry sites, catheter tips and blood were examined for plasmid DNA profiles. In 4 patients the plasmid profiles of the catheter entry site isolates were identical with those of the blood isolates. In the other 3 bacteremic patients plasmid profiles of the catheter entry site and blood isolates were different. No correlation was observed in the plasmid DNA patterns of isolates obtained from catheter tip cultures as compared to the corresponding blood cultures. The blood isolates from bacteremic patients had different plasmid profiles.

Fur and iron transport proteins in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

The Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius causes a fatal septicaemic disease, resembling fulminant meningococcal sepsis, in children. When isolate F3031 was grown under iron-limiting conditions, the presence of several iron-regulated proteins of 38-110 kDa was revealed by electrophoretic analysis and a Fur homologue was shown by immunoblotting. Dot-blot assays and immunoblotting indicated that BPF cells bound human transferrin and contained transferrin-binding proteins in the outer membrane. However, the binding activity and the biosynthesis of these proteins were detected even under iron-rich conditions. Immunoblot analysis demonstrated the presence of a periplasmic protein related to the ferric iron-binding protein A (FbpA), the major iron-binding protein described in Neisseria spp. However, the FbpA homologue in strain F3031 was constitutively expressed and was smaller than the periplasmic protein detected in H. influenzae type b strain Eagan. The periplasm of strain F3031 also contained a protein related to the Streptococcus parasanguis FimA protein which recently has been shown to be involved in iron acquisition in Yersinia pestis. Although the Eagan and F3031 FimA homologues had a similar mol. wt, of 31 kDa, the expression of the BPF fimA-like gene was not regulated by the iron concentration of the culture medium.

Dissemination of plasmid-mediated amikacin resistance among pathogenic Klebsiella pneumoniae.

Plasmids isolated from Klebsiella pneumoniae strains that caused outbreaks in pediatric units in various geographical regions of Argentina harbored genetic determinants for resistance to amikacin. By using restriction endonuclease and Southern blot hybridization analysis it was determined that all of the strains carried plasmids with Tn1331-related elements indicating that transposition of these elements may have played an important role in the dissemination process of resistance to amikacin.

Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing.

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.

Genetics and molecular biology of siderophore-mediated iron transport in bacteria.

The possession of specialized iron transport systems may be crucial for bacteria to override the iron limitation imposed by the host or the environment. One of the most commonly found strategies evolved by microorganisms is the production of siderophores, low-molecular-weight iron chelators that have very high constants of association for their complexes with iron. Thus, siderophores act as extracellular solubilizing agents for iron from minerals or organic compounds, such as transferrin and lactoferrin in the host vertebrate, under conditions of iron limitation. Transport of iron into the cell cytosol is mediated by specific membrane receptor and transport systems which recognize the iron-siderophore complexes. In this review I have analyzed in detail three siderophore-mediated iron uptake systems: the plasmid-encoded anguibactin system of Vibrio anguillarum, the aerobactin-mediated iron assimilation system present in the pColV-K30 plasmid and in the chromosomes of many enteric bacteria, and the chromosomally encoded enterobactin iron uptake system, found in Escherichia coli, Shigella spp., Salmonella spp., and other members of the family Enterobacteriaceae. The siderophore systems encoded by Pseudomonas aeruginosa, namely, pyochelin and pyoverdin, as well as the siderophore amonabactin, specified by Aeromonas hydrophila, are also discussed. The potential role of siderophore-mediated systems as virulence determinants in the specific host-bacteria interaction leading to disease is also analyzed with respect to the influence of these systems in the expression of other factors, such as toxins, in the bacterial virulence repertoire.

A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system.

Many of the high-virulence strains of the marine fish pathogen Vibrio anguillarum isolated from epizootics of the widespread fish disease vibriosis, harbour a specific plamid class which is absent from low-virulence strains. Curing experiments have confirmed a link between this specific plasmic class and the ability of V. anguillarum to establish infections. In general, all bacterial virulence factors promote growth in the antagonistic environment of the host defence mechanisms. One line of defence is provided by the proteins transferrin and lactoferrin, which bind iron, rendering it unavailable to pathogens. A mechanism whereby invading bacteria may successfully compete for the otherwise unavailable iron could therefore become crucial in enabling them to proliferate in body fluids and tissues. I report here evidence which shows that the V. anguillarum virulence plasmid specifies a very efficient iron-sequestering system enabling bacteria to survive in conditions of limited iron availability.

Three origins of replication are active in vivo in the R plasmid RSF1040.

Replicating DNA molecules of RSF1040, a deletion derivative of the conjugative R plasmid R6K, are cleaved at a single site by the Eco RI restriction endonuclease. Microscopic analysis of Eco RI-cleaved RSF1040 replicative intermediates synthesized in vivo indicates that initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma. The relative frequencies of initiations at these three origins are different from those found in vitro.

Divergence of the aerobactin iron uptake systems encoded by plasmids pColV-K30 in Escherichia coli K-12 and pSMN1 in Aerobacter aerogenes 62-1.

Although the aerobactin-mediated iron uptake system has been characterized genetically in Escherichia coli, the siderophore aerobactin was chemically characterized after purification from culture supernatants of Aerobacter aerogenes 62-1, a member of the Klebsielleae. We have cloned and mapped the genes encoding the aerobactin system genes of A. aerogenes 62-1 and begun characterization of the relevant proteins and enzymatic activities of this plasmid-mediated aerobactin system. Published chemical data indicate that the siderophore aerobactin of E. coli is the same molecule as the aerobactin of Aerobacter aerogenes 62-1, but we have found that both the genes and the complement of proteins making up the biosynthetic enzymes in the two systems have diverged. In contrast, the outer membrane receptors for ferric aerobactin of the two systems showed immunologic cross-reactivity, were of the same molecular size (74 kilodaltons), and were encoded by homologous DNA sequences.