Jorge H. Crosa

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.

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

SUMMARY The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.

Iron Transport in Bacteria

Iron Transport in Bacteria, a survey of research conducted over the past 50 years, examines the advances in technology and the recent availability of sequences of microbial genomes that have led to an explosion of knowledge in the field of iron transport systems. Analysis of genomes has identified new systems, and new models for transport have been suggested by crystallography and structural determinations of the membrane transport proteins. Providing an overview of up-to-date information available on iron and microbial virulence, Iron Transport in Bacteria offers insight into development and future directions that will fascinate graduate and advanced undergraduate students and equip instructors in pathogenesis and infectious diseases. The book comprises five concise sections; the first discusses the structures, chemical properties, and biosynthesis of the microbial products, such as siderophores and hemophores used by these organisms to acquire iron. The second section explores the transport of these compounds into gram-negative bacteria. The remaining sections cover iron transport in the prototype, E. coli K-12; iron transport systems in selected pathogenic microorganisms; and iron transport in ecology. Hardcover, 499 pages, full-color insert, illustrations, index.

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 hospitals 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.

Genetic Organization of the Region Encoding Regulation, Biosynthesis, and Transport of Rhizobactin 1021, a Siderophore Produced by Sinorhizobium meliloti

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.

Complete Sequence of Virulence Plasmid pJM1 from the Marine Fish Pathogen Vibrio anguillarum Strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.

The HlyU Protein Is a Positive Regulator of rtxA1, a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii

ABSTRACT We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2β (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host “addiction” genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.

Evidence for the role of a siderophore in promoting Vibrio anguillarum infections

Vibrio anguillarum strain 775 harbouring the virulence plasmid pJM1 produces a plasmid-mediated siderophore that can crossfeed siderophore-deficient, receptor-proficient mutants of V. anguillarum in vitro. Experimental infections of salmonid fishes with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-proficient mutant resulted in recovery of both the wild-type and the mutant strain, while in infections with mixtures consisting of the wild-type strain and a siderophore-deficient, receptor-deficient mutant only the wild-type strain could be recovered. These results suggest that the V. anguillarum plasmid-mediated siderophore is produced in vivo in a diffusible form and that it is an important factor of virulence.

Two tonB Systems Function in Iron Transport in Vibrio anguillarum, but Only One Is Essential for Virulence

ABSTRACT We have identified two functional tonB systems in the marine fish pathogen Vibrio anguillarum, tonB1 and tonB2. Each of the tonB genes is transcribed in an operon with the cognate exbB and exbD genes in response to iron limitation. Only tonB2 is essential for transport of ferric anguibactin and virulence.