Providencia

Carbohydr Res, 2004 Oct 20, 339(15), 2621 - 6
The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47; Ovchinnikova OG et al.; The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51 . Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: {carbohydrate structure: see text}

J Chem Ecol, 2004 Jun, 30(6), 1183 - 201
Pseudopterosin content variability of the purple sea whip Pseudopterogorgia elisabethae at the islands of San Andres and Providencia (SW Caribbean); Puyana M et al.; To determine pseudopterosin composition and concentration in colonies of Pseudopterogorgia elisabethae from the islands of San Andres and Providencia, we collected fragments of individual colonies at various sites and depth ranges around the islands . Chromatographic profiles of the polar fraction, particularly those obtained by HPLC-MS analyses, allowed us to recognize two different chemotypes . Chemotype 1 characterized samples from Providencia whereas chemotype 2 characterized samples from San Andres . A complex pseudopterosin mixture (compounds 1-13) characterized chemotype 1 . These compounds were isolated by a combination of chromatographic methods and identified by spectroscopic methods (MS, UV, 1H, and 13C NMR) . We identified the known pseudopterosins G and K and seco-pseudopterosin A . We also isolated and identified seven new compounds, pseudopterosins P-V, isomers of known pseudopterosins . Pseudopterosins G and K were found at concentrations ranging between 1 and 3% of the animal dry mass . Pseudopterosins Q and U were the major compounds reaching up to 6% of the animal dry mass at some locations . Major metabolites in chemotype 2 had a molecular weight and fragmentation pattern different from that observed in the pseudopterosins, as determined by HPLC-MS . Total pseudopterosin concentration in this chemotype was below 3% dry mass at all sites . Total pseudopterosin concentration was significantly higher in chemotype 1, with concentrations ranging between 4 and 20% dry mass . At most locations on Providencia, however, total pseudopterosin concentration ranged between 11 and 15% dry mass . Concentrations exceed reports from other locations in the Caribbean . Furthermore, pseudopterosin composition in our samples is quite different from those in specimens of P . elisabethae from the Bahamas and Bermuda . Pseudopterosins G, K, and P-V are characteristic of P . elisabethae colonies from the island of Providencia, while pseudopterosins A-D are characteristic of colonies of P . elisabethae from the Bahamas islands, and pseudopterosins E-L have been isolated from P . elisabethae from the Bahamas and Bermuda . The overall morphology of P . elisabethae can be variable, and chemical differences are not correlated to specific morphs . We confirmed the species identity of each colony by morphological and sclerite analysis and found no significant differences in sclerite dimensions among different colonies and chemotypes.

Carbohydr Res, 2004 Jun 22, 339(9), 1655 - 61
Structure of an acidic O-specific polysaccharide from marine bacterium Shewanella fidelis KMM 3582T containing Nepsilon-{(S)-1-carboxyethyl}-Nalpha-(D-galacturonoyl)-L-lysine; Kilcoyne M et al.; The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of the marine bacterium Shewanella fidelis type strain KMM 3582T and studied by sugar analysis along with 1H and 13C NMR spectroscopy including one-dimensional NOE in difference mode and two-dimensional experiments . The polysaccharide was found to consist of linear tetrasaccharide repeating units containing Nepsilon-{(S)-1-carboxyethyl}-Nalpha-(D-galacturonoyl)-L-lysine and having the following structure: {See text.} The amide of D-galacturonic acid with Nepsilon-{(S)-1-carboxyethyl}-L-lysine ('alaninolysine', 2S,8S-AlaLys) was found for the first time in nature as a component of the O-specific polysaccharide of Providencia rustigianii O14 (Carbohydr . Res . 2003, 338, 1009-1016).

Carbohydr Res, 2004 Jun 1, 339(8), 1557 - 60
Structure of the O-polysaccharide of Providencia stuartii O49; Bushmarinov IS et al.; The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments . The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->

J Microbiol Methods, 2004 Jun, 57(3), 409 - 13
Universal primer PCR with DGGE for rapid detection of bacterial pathogens; Ji N et al.; A universal primer PCR (UPPCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens . The results show that this method is efficient at amplifying the conserved regions of bacterial 16S rRNA genes with universal primers and can detect causative bacterial pathogens rapidly . Six species of bacteria from fisheries (Pseudomonas fluorescens, Vibrio anguillarum, Aeromonas hydrophila, Vibrio fluvialis, Providencia rettgeri and Aeromonas sobria) were examined . Our results indicate that the approach we undertook can be adopted not only for axenic bacterial populations but also for mixed communities as well . Furthermore, we were able to achieve the rapid detection of multiple bacteria a single in sample . In addition, UPPCR-DGGE was shown to be better than previously reported UPPCR-single-stranded conformation polymorphism (SSCP)-based methods for the rapid detection of bacterial pathogens.

Mol Microbiol, 2004 May, 52(4), 933 - 45
Cell-to-cell signalling in Escherichia coli and Salmonella enterica; Ahmer BM; Cell-to-cell signalling in prokaryotes that leads to co-ordinated behaviour has been termed quorum sensing . This type of signalling can have profound impacts on microbial community structure and host-microbe interactions . The Gram-negative quorum-sensing systems were first discovered and extensively characterized in the marine Vibrios . Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica . Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal-generating enzyme . Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species . Conversely, E . coli and Salmonella encode the signal-generating component of a second system (a LuxS homologue that generates AI-2), but the sensory apparatus for AI-2 differs substantially from the Vibrio system . The only genes currently known to be regulated by AI-2 in Salmonella encode an active uptake and modification system for AI-2 . Therefore, it is not yet clear whether Salmonella uses AI-2 as a signal molecule or whether AI-2 has some other function . In E . coli, the functions of both SdiA and AI-2 are unclear due to pleiotropy . Genetic strategies to identify novel signalling systems have been performed with E . coli and Providencia stuartii . Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide . The latter system has homologues in E . coli and Salmonella, as well as other bacteria, plants and animals . In fact, the protease components from Providencia and Drosophila are functionally interchangeable.

J Invertebr Pathol, 2004 Jan, 85(1), 9 - 17
Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT; de Oliveira D et al.; Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids . V . zonata specimens were collected on rural properties in Londrina, state of Parana, Southern Brazil . Inoculation of Leptomonas 563DT into V . zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels . Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia . In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V . zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect . The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids . We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages . A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism.

Carbohydr Res, 2004 Jan 22, 339(2), 415 - 9
Structure of the O-polysaccharide of Providencia alcalifaciens O19; Kocharova NA et al.; Studies of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) of Providencia alcalifaciens O19 by sugar and methylation analyses along with NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments, showed that the pentasaccharide repeating unit of the polysaccharide has the following structure: {structure: see text} where Fuc3NAc is 3-acetamido-3,6-dideoxygalactose . The unique structure of the O-antigen and serological data are in consistence with classification of this bacterium in a separate Providencia serogroup.

Carbohydr Res, 2004 Jan 22, 339(2), 195 - 200
Structure of the O-polysaccharide of Providencia stuartii O4 containing 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose; Kocharova NA et al.; The O-polysaccharide of Providencia stuartii O4 was obtained by mild acid degradation of the lipopolysaccharide, and the following structure of the pentasaccharide repeating unit was established: {structure: see text} where D-Qui4N(L-AspAc) is 4-(N-acetyl-L-aspart-4-yl)amino-4,6-dideoxy-D-glucose, which has not been hitherto found in bacterial polysaccharides . Structural studies were performed using sugar and methylation analyses, Smith degradation and NMR spectroscopy, including conventional 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments as well as COSY and NOESY experiments run in an H(2)O-D(2)O mixture to reveal correlations for NH protons.

J Antimicrob Chemother, 2004 Feb, 53(2), 277 - 82 Epub 2003 Dec 19.
ESBL-producing multidrug-resistant Providencia stuartii infections in a university hospital; Tumbarello M et al.; OBJECTIVES: To investigate the epidemiological and clinical findings of extended-spectrum beta-lactamase (ESBL)-producing Providencia stuartii infections in a large Italian university hospital . PATIENTS AND METHODS: All consecutive episodes of P . stuartii infection that occurred during 1999-2002 were included in the study . For each patient, we recorded the area of hospitalization and drug susceptibility of the P . stuartii strains . Patients with ESBL-producing P . stuartii infection were considered cases and those with non-ESBL-producing P . stuartii infection were used as controls . RESULTS: One hundred and sixteen (52%) out of 223 P . stuartii strains collected during the study period were found to be ESBL-producing . On the basis of PCR and DNA sequencing experiments, TEM-52 was identified in 87% of isolates and TEM-72 in 13% . All ESBL-producing P . stuartii infections were nosocomially acquired . The prevalence increased from 31% of P . stuartii infections in 1999 to 62% in 2002 (P = 0.04) . All 116 strains were classified as ESBL-producing multidrug-resistant P . stuartii, since 88% of the isolates were cross-resistant to ciprofloxacin and amikacin and the other 12% were cross-resistant to ciprofloxacin and gentamicin . At logistic regression analysis, advanced age (P < 0.001), previous hospitalization (P < 0.01), neoplastic disease (P < 0.001) and previous antibiotic therapy (P < 0.001) were independent risk factors for the development of ESBL-producing infections . CONCLUSIONS: This 4 year surveillance of Providencia complaints clearly indicates that infections caused by ESBL-producing multidrug-resistant P . stuartii are an emerging problem.

J Med Microbiol, 2003 Aug, 52(Pt 8), 633 - 6
Providencia alcalifaciens strains translocate from the gastrointestinal tract and are resistant to lytic activity of serum complement; Vieira AB et al.; The ability of Providencia alcalifaciens strains, isolated from patients with diarrhoeal disease, to translocate from the gastrointestinal tract and their resistance to serum complement lytic activity were investigated and compared with previously characterized differential invasive capabilities in HeLa cells . Translocation ability to several extraintestinal sites and resistance to lysis by human serum complement were observed in both highly invasive and non-invasive strains . These characteristics have not been previously described in P . alcalifaciens and their potential role in causing disseminated infections should therefore be considered.

Mol Cell, 2003 Jun, 11(6), 1425 - 34
Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain; Urban S et al.; Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors . We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD) . This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices . Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy . We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases . Intramembrane cleavage of these proteins is required for host cell invasion . These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jun, 35(6), 573 - 9
Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling; Zhou Z et al.; Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method . The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9% . The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries . By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E . coli JM109(DE3) . Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently . It was shown that the primary structures of selected positives exhibited significant diversity among each library . The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA . It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis . Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.

J Antimicrob Chemother, 2003 Apr, 51(4), 787 - 90 Epub 2003 Mar 13.
An integron cassette encoding erythromycin esterase, ere(A), from Providencia stuartii; Plante I et al.; We have mapped the variable region of the two class 1 integrons found in the multiresistant strain Providencia stuartii 1723 . Integron 1 contains a new arrangement of gene cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides used for clinical treatment . Integron 2 contains a variant of the gene cassette ere(A), coding for an erythromycin esterase, whose nucleotide sequence shares 93.7% DNA identity with ere(A) from Escherichia coli BM2195 plasmid pIP1100.

Genome Biol . 2003;4(3):R19 . Epub 2003 Feb 28.
The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers; Koonin EV et al.; BACKGROUND: The rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins . They are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes . On the basis of experimental studies with the developmental regulator rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes . RESULTS: Phylogenetic tree analysis carried out using several independent methods for tree constructions and the corresponding statistical tests suggests that, despite its broad distribution in all three superkingdoms, the rhomboid family was not present in the last universal common ancestor of extant life forms . Instead, we propose that rhomboids evolved in bacteria and have been acquired by archaea and eukaryotes through several independent horizontal gene transfers . In eukaryotes, two distinct, ancient acquisitions apparently gave rise to the two major subfamilies, typified by rhomboid and PARL (presenilins-associated rhomboid-like protein), respectively . Subsequent evolution of the rhomboid family in eukaryotes proceeded by multiple duplications and functional diversification through the addition of extra transmembrane helices and other domains in different orientations relative to the conserved core that harbors the protease activity . CONCLUSIONS: Although the near-universal presence of the rhomboid family in bacteria, archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor, phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer . This emphasizes the importance of explicit phylogenetic analysis for the reconstruction of ancestral life forms . A hypothetical scenario for the origin of intracellular membrane proteases from membrane transporters is proposed.

Rev Med Chil, 2002 Dec, 130(12), 1335 - 42
{Nutrients intake in elderly people living in Providence, Santiago de Chile}; Castillo O et al.; BACKGROUND: The information available on food intake in the elderly in Chile is restricted to individuals of low socioeconomic groups, but there is no data available on food intake in elderly of higher income groups . AIM: To assess food intake in a group of elderly people from Providencia County in Santiago, a middle income community . SUBJECTS AND METHODS: Forty one subjects (20 male), aged 60 to 73 years, were studied . Trained volunteers applied a 3 days food registry, to determine food intake . Intake was assessed using 1985 FAO/OMS/UNU recommendations for energy intake and USA Food and Nutrition Board recommendations for micronutrient intake . RESULTS: The studied subjects had an adequate macronutrient intake, when compared with current recommendations . There was a relatively low intake of calories from fat (24.6% in males and 26.1% in females) . Also, vitamin and mineral intake was adequate with the exception of calcium (64.5% and 57.9% of recommendation in males and in females respectively), zinc and folic acid (74.2% and 62.4% in males and females respectively) . The intake of legumes and cereals was relatively low . CONCLUSIONS: Food intake in this group of individuals was substantially higher than that reported previously for poor elderly Chileans and similar to that of industrialized countries . Food intake of the elderly is probably related to socioeconomic level.

Cell Mol Life Sci, 2002 Dec, 59(12), 2065 - 70
Comparison of SXT and R391, two conjugative integrating elements: definition of a genetic backbone for the mobilization of resistance determinants; Beaber JW et al.; The SXT element (SXT) is becoming an increasingly prevalent vector for the dissemination of antibiotic resistances in Vibrio cholerae . SXT is a member of a larger family of elements, formerly defined as IncJ plasmids, that are self-transmissible by conjugation and integrate site-specifically into the host chromosome . Comparison of the DNA sequences of SXT and R391, an IncJ element from Providencia rettgeri, indicate that these elements consist of a conserved backbone that mediates the regulation, excision/integration and conjugative transfer of the elements . Both elements have insertions into this backbone that either confer the element-specific properties or are of unknown function . Interestingly, the conserved SXT and R391 backbone apparently contains hotspots for insertion of additional DNA sequences . This backbone represents a scaffold for the mobilization of genetic material between a wide range of gram-negative bacteria, allowing for rapid adaptation to changing environments.

J Zoo Wildl Med, 2002 Dec, 33(4), 301 - 10
Osteomyelitis associated with Salmonella enterica SS arizonae in a colony of ridgenose rattlesnakes (Crotalus willardi); Ramsay EC et al.; The identification of three Arizona ridgenose rattlesnakes (Crotalus willardi) with Salmonella arizonae-associated osteomyelitis led to a 5-yr prospective study of radiographic signs and Salmonella intestinal carriage rates in a 19-member colony of this rattlesnake species . Ventrodorsal radiographs were performed and cloacal swabs were cultured for Salmonella spp . annually . Ten snakes survived the 5-yr period, with six of them remaining free of bony lesions . Three snakes that had no bony lesions in 1995 developed radiographic signs of osteomyelitis during the study . Six snakes with bony lesions at the beginning of the study died or were euthanatized due to osteomyelitis during the study . The radiographic signs of osteomyelitis were progressive for five snakes that were serially radiographed . Only one snake with radiographic signs of osteomyelitis at the beginning of the study was still alive at the end of the study, and this animal's bony lesions were more extensive at the end . Thirty-nine intestinal S . arizonae isolates, representing 13 serotypes, were obtained from the 19 snakes . Salmonella arizonae serotype 56:Z4,Z23 was isolated only once from a cloacal culture, from a snake that had no radiographic bone lesions . Twelve extraintestinal Salmonella isolates, representing two serotypes, were isolated from six snakes . All extraintestinal isolates except one were of S . arizonae serotype 56:Z4,Z23, and all isolates from bone were of this serotype . One snake with characteristic bone lesions died, and Providencia rettgeri was cultured from each of the tissues cultured, whereas no Salmonella spp . were isolated from this snake . Salmonella arizonae serotype 56:Z4,Z23 {corrected} appears to have a tropism for bone and other extraintestinal sites in C . willardi and may cause a progressive, ultimately fatal disease in this species.

Carbohydr Res, 2002 Oct 8, 337(18), 1667 - 71
Structure of the O-specific polysaccharide of Providencia alcalifaciens O16 containing N-acetylmuramic acid; Kocharova NA et al.; The O-specific polysaccharide of Providencia alcalifaciens O16 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments . It was found that the polysaccharide contains N-acetylmuramic acid, which was isolated by solvolysis with trifluoromethanesulfonic acid and identified by the specific optical rotation and NMR spectroscopy . The following structure of the trisaccharide repeating-unit of the polysaccharide was established:

Infection, 2002 Oct, 30(5), 306 - 9
Clustering of bloodstream infections during maggot debridement therapy using contaminated larvae of Protophormia terraenovae; Nuesch R et al.; BACKGROUND: The treatment of human wounds with fly larvae is an ancient procedure recently reintroduced into medical practice under the term of biosurgery . The crucial technical problem of biosurgery is asepsis of the larvae . PATIENTS AND METHODS: Since February 1999, we conducted a prospective observational study on the use of maggot debridement therapy in the management of ulcers refractory to standard treatment . RESULTS: During the first 5 months we observed five bloodstream infections (four with Providencia stuartii and one with Candida albicans) in 24 patients (21%) treated with maggots . The blood isolates could be traced back to contaminated maggots . Accordingly, the disinfecting procedure of the maggots was optimized and the fly species was changed from Protophormia terraenovae to Phaenicia (Lucilia) sericata . With the new procedure, no case of sepsis occurred in 45 patients treated between January 2000 and December 2001 (p < 0.005) . CONCLUSION: Despite promising benefits, maggot debridement therapy can be threatened by serious infectious complications . With an appropriate disinfecting procedure, maggots free of pathogens can be obtained . Provided the maggots have been effectively disinfected, their application on chronic ulcers seems to be safe.

Curr Biol, 2002 Sep 3, 12(17), 1507 - 12
Conservation of intramembrane proteolytic activity and substrate specificity in prokaryotic and eukaryotic rhomboids; Urban S et al.; Rhomboid is an intramembrane serine protease responsible for the proteolytic activation of Drosophila epidermal growth factor receptor (EGFR) ligands . Although nothing is known about the function of the approximately 100 currently known rhomboid genes conserved throughout evolution, a recent analysis suggests that a Rhomboid from the pathogenic bacterium Providencia stuartii is involved in the production of a quorum-sensing factor . This suggests that an intercellular signaling mechanism may have been conserved between prokaryotes and metazoans . However, the function of prokaryotic Rhomboids is unknown . We have examined the ability of eight prokaryotic Rhomboids to cleave the three Drosophila EGFR ligands . Despite their striking sequence divergence, Rhomboids from one Gram-positive and four Gram-negative species, including Providencia, specifically cleaved Drosophila substrates, but not similar proteins such as Transforming Growth Factor alpha (TGFalpha) and Delta . Although the sequence similarity between these divergent Rhomboids is very limited, all contain the putative serine catalytic triad residues, and their specific mutation abolished protease activity . Therefore, despite low overall homology, the Rhomboids are a family of ancient, functionally conserved intramembrane serine proteases, some of which also have conserved substrate specificity . Moreover, a function for Rhomboids in activating intercellular signaling appears to have evolved early.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12208 - 13 Epub 2002 Sep 09.
A conserved mechanism for extracellular signaling in eukaryotes and prokaryotes; Gallio M et al.; Epidermal growth factor receptor (EGFr) is a key mediator of cell communication during animal development and homeostasis . In Drosophila, the signaling event is commonly regulated by the polytopic membrane protein Rhomboid (RHO), which mediates the proteolytic activation of EGFr ligands, allowing the secretion of the active signal . Until very recently, the biochemical function of RHO had remained elusive . It is now believed that Drosophila RHO is the founder member of a previously undescribed family of serine proteases, and that it could be directly responsible for the unusual, intramembranous cleavage of EGFr ligands . Here we show that the function of RHO is conserved in Gram-negative bacteria . AarA, a Providencia stuartii RHO-related protein, is active in Drosophila on the fly EGFr ligands . Vice versa, Drosophila RHO-1 can effectively rescue the bacterium's ability to produce or release the signal that activates density-dependent gene regulation (or quorum sensing) . This study provides the first evidence that prokaryotic and eukaryotic RHOs could have a conserved role in cell communication and that their biochemical properties could be more similar than previously anticipated.

J Bacteriol, 2002 Sep, 184(18), 5158 - 69
R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements; Boltner D et al.; The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements . Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair . While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139 . Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements . A total of 96 open reading frames were identified; of these, 30 could not be assigned a function . Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27 . A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified . Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

J Med Microbiol, 2002 Aug, 51(8), 682 - 6
TnphoA mutants of Providencia alcalifaciens with altered invasiveness of HEp-2 cells; Rahman M et al.; Recent studies have shown that Providencia alcalifaciens is a diarrhoeal pathogen . It may cause diarrhoea by an invasive mechanism, as it invades cultured mammalian cells in vitro and intestinal epithelial cells of experimentally inoculated rabbits in vivo . To locate the gene(s) involved in invasion, TnphoA mutants of a diarrhoeal isolate of P . alcalifaciens were generated . Compared with the parent strain, these mutants exhibited negligible invasion and actin condensation in HEp-2 cells . TnphoA insertion was located in fragments of 4.9 kb and 11.1 kb of the bacterial chromosome by Southern blot . These mutants did not secrete a 28-kDa protein, which may be involved in invasion . It should be possible now to study the gene(s) involved in invasion of P . alcalifaciens with these mutants . This investigation is another example of the usefulness of TnphoA mutagenesis in the study of bacterial virulence genes.

Biotechnol Prog, 2002 May-Jun, 18(3), 668 - 71
Effect of pH on high-temperature production of bacterial penicillin acylase in Escherichia coli; Huang SW et al.; High-temperature-oriented production of bacterial penicillin acylase (PAC), which is usually expressed at low temperatures (less than 30 degrees C), was demonstrated in this study via heterologous expression of the Providencia rettgeri (P . rettgeri) pac gene in Escherichia coli (E . coli) . While it is possible to produce PAC at a temperature as high as 37 degrees C, the environmental condition (specifically, culture pH) critically affected culture performance . Production of PAC at 37 degrees C was feasible only when culture pH was close to neutral (i.e., 6.5-7.5) . Outside this pH range, cell physiology for the host/vector system was seriously affected, resulting in poor culture performance . In acidic culture environments, temperature significantly affected the pac expression level and specific PAC activity decreased with an increase in culture temperature . In basic culture environments, cell growth was seriously inhibited though the pac expression level was minimally affected by temperature . Such unusual types of pH and temperature effects on pac expression were never reported for bacterial PACs . The results suggest that culture pH should be precisely controlled for the current host/vector systems being applied on the overproduction of P . rettgeri PAC in E . coli at high temperatures.

Biotechnol Prog, 2002 Mar-Apr, 18(2), 330 - 6
High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization; Ljubijankic G et al.; Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae . Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion . The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted . Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme . The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters . The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.

Curr Microbiol, 2001 Dec, 43(6), 414 - 7
Non-invasive Providencia alcalifaciens strains fail to attach to HEp-2 cells; Khashe S et al.; We investigated the adherence properties of six P . alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells . Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection . In contrast, weakly or non-invasive P . alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection . Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E . coli (EPEC) . These results suggest that the strain variation in the ability of P . alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates.

Rev Neurol, 2001 Jul 1-15, 33(1), 82 - 9
{Antoniana Margarita: Gómez Pereira, Francisco Lobato and the antecedents of cerebral mechanicism during the Spanish renaissance}; Martin-Araguz A et al.; The year 2000 is the fifth century of the birth, in Medina del Campo (Vallodolid, Spain) of licenciado Perea (Gomez Perea or Pereira) . A man of the Renaissance, he was an outstanding doctor, humanist, theologist, nominalist philosopher, naturalist and practical engineer . He developed the first modern theory of behavior, based purely on mechanicistic principles, describing his ideas in a text known by the curious title of Antoniana Margarita . The objective of this paper is to pay him homage on the fifth centenary of his birth, making a historiographic study of Gomez Perea and his works, with particular emphasis on the ideological basis and its relationship with Renaissance hydraulic engineering, collaborating with his colleague Francisco Lobato, author of one of the only two pretechnological codices of sixteenth century Spain . The book Antoniana Margarita is written in Renaissance Latin and was published in Medina del Campo in 1554 . It represents the first truly modern approach to brain function which excludes the providencialist concepts of Galen involving the soul and the spirit, in vogue until then, transmitted through the Arab and Scholastic tradition . Analyzing his theory of the automatism of animals Perea made the first description ever of the reflex arc and the conditioned reflex . He also established a topographical model of the brain in which he sketched the functioning of the prefrontal cortex and neurophysiology of memory . Perea was the immediate forerunner of Neuropsychology and of the methodology and organicist thought which predominates in modern Neurobiology . He was also a visionary of the Evolution of Darwin and of modern aetiology.

Rev Biol Trop, 2000 Dec, 48(4), 883 - 96
{Reef fishes community structure in 4 atolls of the San Andrés - Providencia Archipelago (Southwestern Caribbean)}; Mejia LS et al.; In 1994 and 1995, 131 visual censuses of reef fishes were made using the stationary sampling method in Courtown, Albuquerque, Serrana and Roncador, four atolls of the Archipelago of San Andres and Old Providence in the Southwestern Caribbean . Fish species and their abundances were recorded in four geomorphologic zones: lagoon, windward barrier reef, windward terrace and forereef terrace . A total of 98 species were censused; the most abundant were Chromis cyanea (14%), Clepticus parra (14%) and Stegastes partitus (10%) . The most abundant families were Pomacentridae (37%), Labridae (28%) and Scaridae (10%) . Analysis of similarities showed that differences between zones were greater than differences between atolls, but lagoon and forereef terrace were not significantly different . Cluster and ordination analysis confirmed these results; in addition, the ordination analysis placed the groups according to depth and wave-exposure gradients, suggesting that these two physical variables were responsibles for the clustering . Differences in equitability and species richness appear also due to these variables . Inverse analysis showed in each group few characteristic species, then the differences between zones were due specially to dominance of some species . The dominant trophic categories were planktivorous and herbivorous that were significantly different between zones . In shallow zones (shallow lagoonal patch reefs) and high wave-exposed zones (winward barrier reef) dominated herbivorous fishes, while in deeper zones (terraces and deep lagoonal patch reefs) planktivorous were more abundant.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2238 - 44
Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii; Franklin K et al.; The gene coding for aminoglycoside 2'-N-acetyltransferase Ia {AAC(2')-Ia} from Providencia stuartii was amplified by PCR and cloned . The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag . The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75 . Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity . The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1 . The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM . Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest . Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites . No correlation was found between these kinetic parameters and MICs determined for P . stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.

FEMS Microbiol Lett, 2001 Mar 1, 196(1), 25 - 9
Role of SspA in the density-dependent expression of the transcriptional activator AarP in Providencia stuartii; Ding X et al.; The AarP protein in Providencia stuartii encodes a small transcriptional activator which activates the chromosomal aminoglycoside acetyltransferase aac(2')-Ia gene . In addition, AarP activates genes involved in a multiple antibiotic resistance (Mar) phenotype . Expression of an aarP-lacZ fusion increased in a density-dependent manner and reached peak levels at stationary phase . The expression of an aarP-lacZ fusion could be prematurely activated in cells at early to mid-exponential phase by the addition of spent culture supernatants from stationary phase cultures or by ethyl acetate extracts of these supernatants . Nutrient starvation had a negligible effect on aarP expression . In a search for mutations that block aarP activation at stationary phase, a mini-Tn5Cm insertion has been identified within a gene whose product was 77% identical to SspA, a regulatory protein involved in stationary phase gene expression and virulence . An unmarked sspA null allele (sspA2) was created by allelic replacement to further examine the role of sspA in P . stuartii . The sspA2 allele resulted in substantial decrease in aarP mRNA accumulation at various phases of growth . Furthermore, in an sspA mutant background, the aarP-lacZ fusion was no longer activated by an extracellular signal.

J Bacteriol, 2001 Feb, 183(4), 1124 - 32
Formation of chromosomal tandem arrays of the SXT element and R391, two conjugative chromosomally integrating elements that share an attachment site; Hochhut B et al.; The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a Vibrio cholerae O139 isolate from India, and IncJ element R391, originally derived from a South African Providencia rettgeri isolate, were found to be genetically and functionally related . Both of these constins integrate site specifically into the Escherichia coli chromosome at an identical attachment site within the 5' end of prfC . They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar tra genes . Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination . The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants . In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays . Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element . In these assays, R391 was found to have a stronger effect on SXT stability than vice versa . The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.

J Med Entomol, 2000 Nov, 37(6), 924 - 8
Diversity and contribution of the intestinal bacterial community to the development of Musca domestica (Diptera: Muscidae) larvae; Zurek L et al.; The bacterial diversity in the intestinal tract of Musca domestica L . was examined in larvae collected from turkey bedding and corn silage . Aerobic culturing yielded 25 bacterial species, including 11 from larvae collected from turkey bedding and 14 from larvae collected from corn silage . Providencia rettgeri (Hadley, Elkins & Caldwell) was the only species common to both environments . Two mammalian pathogens, Yersinia pseudotuberculosis (Pfeiffer) and Ochrobactrum anthropi (Holmes), were isolated from the larval intestinal tracts . The majority of isolates represented facultatively anaerobic heterotrophs capable of fermentation . The significance of these bacteria for development of house fly larvae was evaluated by bioassays on trypticase soy egg yolk agar . Pure cultures of individual bacterial species isolated from the intestinal tract of larvae from turkey bedding supported development of flies to a much greater extent than those isolated from larvae from corn silage . House fly development was best supported by a Streptococcus sanguis (White) isolate . The significance of bacteria for development of house flies is discussed.

J Bacteriol, 2000 Sep, 182(18), 5139 - 46
Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis; Poon WW et al.; It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis . Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis . Both the P . stuartii aarF and E . coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol . Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E . coli strain AN59, which contains the ubiB409 mutant allele . Analysis of the mutation in the E . coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene . The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB . The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene . AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes . Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination . In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis . The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member . The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.

Monaldi Arch Chest Dis, 2000 Apr, 55(2), 110 - 3
HHV-8 is not a cofactor in the pathogenesis of environmentally induced malignant pleural mesothelioma; Olut AI et al.; After the recognition of human herpes virus 8 (HHV-8) in Kaposi's sarcoma lesions, this new virus has been shown to be associated with various types of malignancy . One of them, body cavity-based lymphoma, is a high grade B-cell lymphoma arising from the body cavities . Similarly, mesothelioma is a tumour that originates from the serosal linings of the pleural, pericardial and peritoneal cavities . One of the striking characteristics of mesothelioma cells is the secretion of interleukin-6 (IL-6) . Also, it is known that HHV-8 upregulates the levels of IL-6, and this virally originated IL-6 is a well-established growth factor for HHV-8-associated lesions . Therefore, it was hypothesized that HHV-8 may have a role in the pathogenesis of malignant mesothelioma . Twenty-nine pleural biopsy specimens from environmentally induced malignant mesothelioma patients were investigated for the presence of HHV-8 deoxyribonucleic acid (DNA) using the polymerase chain reaction (PCR) . Control pleural samples were collected from 15 biopsy specimens from patients with tuberculosis . From all samples, a segment of the beta-globulin gene was amplified in order to make sure that the DNA was extracted properly and did not contain any inhibitors . The specificity of the PCR amplification was confirmed by means of restriction enzyme analysis using Providencia stuartii I . PCR did not reveal HHV-8 DNA in any of the mesothelioma patients or in the control group . It was possible to amplify a segment of the human beta-globulin gene from all the samples of the patient and control groups . HHV-8 DNA was amplified in the control sample, which was a tissue biopsy specimen from a Kaposi's sarcoma lesion, and it was confirmed that the amplified DNA belonged to HHV-8 by restriction enzyme analysis . Malignant mesothelioma continues to be a public health problem in rural parts of Anatolia, Turkey . The major causal factor of the disease is exposure to asbestos and fibrous zeolite (erionite) . It seems that there must be some aetiological factors other than exposure to these minerals as not all patients exposed to asbestos develop the disease and the disease is not always associated with any known exposure . From the present study, it was concluded that human herpes virus 8 does not seem to be associated with environmentally induced malignant mesothelioma in Turkey . Other possible causal factors of malignant mesothelioma should be sought.

Biochemistry (Mosc), 2000 Jun, 65(6), 677 - 84
Structure of an acidic O-specific polysaccharide of the bacterium Providencia alcalifaciens O7; Bystrova OV et al.; An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O7 and purified by gel chromatography followed by anion-exchange chromatography . On the basis of full acid hydrolysis, methylation, carboxyl reduction, selective cleavage with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional 1H,1H homonuclear and H-detected 1H,13C heteronuclear correlation spectroscopy and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: {figure}, where Rhap2Ac is 2-O-acetylrhamnopyranose.

Biotechnol Prog, 2000 May-Jun, 16(3), 315 - 8
An approach for enhancing heterologous production of Providencia rettgeri penicillin acylase in Escherichia coli; Chou CP et al.; Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized in Escherichia coli . Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction . The optimum culture conditions for the enzyme production vary for different host/vector systems . With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P . rettgeri . The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system . To our knowledge, this is the first evidence demonstrating the limiting step for the production of P . rettgeri PAC and the existence of the P . rettgeri PAC precursor.

J Bacteriol, 1999 Dec, 181(23), 7185 - 91
Providencia stuartii genes activated by cell-to-cell signaling and identification of a gene required for production or activity of an extracellular factor; Rather PN et al.; By utilizing reporter transposons, five Providencia stuartii genes that are activated by the accumulation of self-produced extracellular signals have been identified . These genes have been designated cma for conditioned medium activated . The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold . Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the cma fusions . In addition, depending on the reporter fusion, peak activity was found in different fractions . The partially purified factors activated in a dose-dependent manner . Characterization of the factors activating the cma fusions indicated that they were stable to heat, alkali, and acid . Furthermore, for each cma fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or alpha-ketoglutarate . The identities of three cma genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import . To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus, aarA, that is required for the activation of four cma fusions . The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.

Protein Sci, 1999 Oct, 8(10), 1971 - 81
Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri; McDonough MA et al.; Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections . Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure . One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source . Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexanamide . The X-ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site-directed mutants with desired specificities . In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 A resolution by molecular replacement . The R-factor after refinement is 0.154 and R-free is 0.165 . Of the 758 residues in the Bro1 penicillin acylase heterodimer (alpha-subunit, 205; beta-subunit, 553), all but the eight C-terminal residues of the alpha-subunit have been modeled based on a partial Bro1 sequence and the complete wild-type P . rettgeri sequence . A tightly bound calcium ion coordinated by one residue from the alpha-subunit and five residues from the beta-subunit has been identified . This enzyme belongs to the superfamily of Ntn hydrolases and uses Ogamma of Ser beta1 as the characteristic N-terminal nucleophile . A mutation of the wild-type Met alpha140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase . The electron density for the N-terminal Gln of the alpha-subunit is best modeled by the cyclized pyroglutamate form . Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P . rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged beta-lactam R-groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi-synthetic cephalosporins.

J Clin Microbiol, 1999 Sep, 37(9), 3048 - 50
Isolation of Providencia heimbachae from human feces; Mohr O'Hara C et al.; Providencia heimbachae was first described in 1986 . It has been isolated from penguin feces and an aborted bovine fetus . To date, there has been no reported isolation of this organism from human specimens . We now report the isolation of P . heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea . The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P . heimbachae.

Antimicrob Agents Chemother, 1999 Jul, 43(7), 1769 - 72
Activation of the 2'-N-acetyltransferase gene {aac(2')-Ia} in Providencia stuartii by an interaction of AarP with the promoter region; Macinga DR et al.; The aac(2')-Ia gene in Providencia stuartii encodes a 2'-N-acetyltransferase capable of acetylating both peptidoglycan and certain aminoglycoside antibiotics . Regulation of the aac(2')-Ia gene is influenced in a positive manner by the product of the aarP gene, which encodes a small transcriptional activator of the AraC (XylS) family . In this study, we demonstrate the sequence requirements at the aac(2')-Ia promoter for AarP binding and activation.

Biochemistry (Mosc), 1999 May, 64(5), 523 - 7
Structure of a neutral O-specific polysaccharide of the bacterium Providencia alcalifaciens O5; Zatonsky GV et al.; A neutral polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) in the ratios 2:1:1 was obtained by mild acid degradation of lipopolysaccharide of the bacterium Providencia alcalifaciens O5 followed by gel chromatography and ion-exchange chromatography or treatment with anhydrous hydrogen fluoride . On the basis of full acid hydrolysis, methylation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), H-detected heteronuclear 1H,13C single-quantum coherence (HSQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established:

Gene, 1999 Mar 4, 228(1-2), 225 - 32
Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae; Ljubijankic G et al.; The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae . Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm . Contrary to the P . rettgeri PAC-encoding gene, the E . coli pac is poorly expressed in yeast . The highest yield of P . rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter . This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system . The recombinant P . rettgeri enzyme is only partially and selectively O-glycosylated . Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all . N-Glycosylation has not been detected.

J Med Microbiol, 1999 Feb, 48(2), 205 - 9
Clonal structure of Providencia alcalifaciens strains isolated from diarrhoeal stools in São Paulo, Brazil; Guth BE et al.; Clonal analysis based on ribotyping demonstrated that Providencia alcalifaciens strains isolated mainly from diarrhoeal stools in Sao Paulo, Brazil, were clustered into two main groups . Eleven distinct ribotype patterns were identified with ClAI, EcoRV and MluI restriction endonucleases . P . alcalifaciens strains with invasive properties were of two ribotype patterns that differed from those identified among non-invasive strains . The ribotyping results confirmed that P . alcalifaciens strains associated with diarrhoeal disease in Sao Paulo represent distinct groups of strains . Although the invasive strains were isolated from different patients over an extended period they were clustered into two genetically related clones, which seemed to be distributed endemically in the population studied.

Front Biosci, 1999 Feb 01, 4, D132 - 40
The chromosomal 2'-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation; Macinga DR et al.; Intrinsic chromosomal acetyltransferases involved in aminoglycoside resistance have been identified in a number of bacteria . In Providencia stuartii, a chromosomal acetyltransferase (AAC(2')-Ia) has been characterized in detail . In addition to the ability to acetylate aminoglycosides, the AAC(2')-Ia enzyme has at least one physiological function, which is the acetylation of peptidoglycan . This modification is likely to influence the autolytic system in P . stuartii . The regulation of aac(2')-Ia expression is extremely complex involving at least seven regulatory genes acting in at least two pathways . This complexity in regulation indicates that aac(2')-Ia expression must be tightly controlled in response to different environmental conditions . This presumably reflects the importance of maintaining correct levels of peptidoglycan acetylation . In this review, a summary of data will be presented involving both the physiological and genetic aspects of aac(2')-Ia in P . stuartii.

Carbohydr Res, 1998 Jun, 309(1), 131 - 3
Identification of N epsilon-{(R)-1-carboxyethyl}-L-lysine in, and the complete structure of, the repeating unit of the O-specific polysaccharide of Providencia alcalifaciens O23; Kocharova NA et al.; N epsilon-{(R)-1-Carboxyethyl}-L-lysine was released by acid hydrolysis from the O-specific polysaccharide of Providencia alcalifaciens O23 and identified by 1H and 13C NMR spectroscopy, GLC-MS after conversion to a di-N-acetylated dimethyl ester, and by comparison with the authentic sample . Solvolysis of the polysaccharide with anhydrous HF resulted in an amide of D-glucuronic acid with N epsilon-{(R)-1-carboxyethyl}-L-lysine . These and published data allowed the determination of the full structure of the repeating unit of the O-specific polysaccharide.

Curr Microbiol, 1998 Sep, 37(3), 159 - 65
Invasion of HEp-2 and other eukaryotic cell lines by Providenciae: further evidence supporting the role of Providencia alcalifaciens in bacterial gastroenteritis; Janda JM et al.; Wild-type strains of Providencia species were evaluated for their ability to invade HEp-2 monolayers based upon microscopic and semi-quantitative assays . Of 14 P . alcalifaciens strains tested, 3 (17%) were found to be highly invasive, 4 (22%) moderately invasive, and the remaining 61% weakly or noninvasive . HEp-2 invasion results were confirmed by thin-section electron microscopy . Invasive capabilities of P . alcalifaciens were greater at higher MOIs (100 to 1000) than at lower inocula (<10 MOI) . No strain of P . stuartii or P . rettgeri tested invaded HEp-2 cells . Quantitative assays of Triton X-100-lysed, HEp-2-invaded cells indicated that between 0.001% and 0 . 013% of the initial bacterial inoculum was gentamicin resistant . Further testing of select strains on various cell lines indicated the efficiency of invasion was Vero > Y1 > INT-407 > HEp-2 . Two isolates recovered from a father and son with prolonged diarrhea after returning from Mexico were found to be identical on the basis of biotype, serotype, and genotype . These results provide additional evidence that some P . alcalifaciens strains cause gastroenteritis.

Mol Microbiol, 1998 Jun, 28(6), 1345 - 53
A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii; Rather PN et al.; A recessive mutation, aarG1, has been identified that resulted in an 18-fold increase in the expression of beta-galactosidase from an aac(2')-lacZ fusion . Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2')-Ia . The effects of aarG1 on aac(2')-Ia expression were mediated by aarP-dependent and -independent mechanisms . The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance . This Mar phenotype also resulted from aarP-dependent and -independent mechanisms . Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem . The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ . The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase . The AarG product was functionally similar to PhoQ, as it was able to restore wild-type levels of maganin resistance to a Salmonella typhimurium phoQ mutant . However, expression of the aarP and aac(2')-Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.

Antimicrob Agents Chemother, 1998 Jun, 42(6), 1459 - 62
Ceftazidime and aztreonam resistance in Providencia stuartii: characterization of a natural TEM-derived extended-spectrum beta-lactamase, TEM-60; Franceschini N et al.; A plasmid-encoded beta-lactamase produced from a clinical strain of Providencia stuartii has been purified and characterized . The gene coding for the beta-lactamase was cloned and sequenced . It appears to be a new natural TEM-derived enzyme, named TEM-60 . Point mutations (Q39K, L51P, E104K, and R164S) are present with respect to the TEM-1 enzyme; the mutation L51P has never been previously reported, with the exception of the chromosomally encoded extended-spectrum beta-lactamase PER-1 . Kinetic parameters relative to penicillins, cephalosporins, and monobactams other than mechanism-based inactivators were related to the in vitro susceptibility phenotype.

J Clin Microbiol, 1998 May, 36(5), 1433 - 5
Association of Providencia alcalifaciens with diarrhea in children; Albert MJ et al.; It has been demonstrated in previous studies that Providencia alcalifaciens can produce diarrhea by an invasive mechanism . In the present study, P . alcalifaciens was isolated from the stool specimens of 17 of 814 diarrheal children younger than 5 years of age (2.1%) and from those of 4 of 814 matched controls (0.49%) (P = 0.004), indicating that the organism is significantly associated with diarrhea . However, 71% of P . alcalifaciens-positive diarrheal children had simultaneous infections with other recognized enteric pathogens.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 959 - 62
Mutations in aarE, the ubiA homolog of Providencia stuartii, result in high-level aminoglycoside resistance and reduced expression of the chromosomal aminoglycoside 2'-N-acetyltransferase; Paradise MR et al.; The aarE1 allele was identified on the basis of the resulting phenotype of increased aminoglycoside resistance . The aarE1 mutation also resulted in a small-colony phenotype and decreased levels of aac(2')-Ia mRNA . The deduced AarE gene product displayed 61% amino acid identity to the Escherichia coli UbiA protein, an octaprenyltransferase required for the second step of ubiquinone biosynthesis . Complementation experiments in both Providencia stuartii and E . coli demonstrated that aarE and ubiA are functionally equivalent.

J Bacteriol, 1998 Jan, 180(1), 128 - 35
Identification and characterization of aarF, a locus required for production of ubiquinone in Providencia stuartii and Escherichia coli and for expression of 2'-N-acetyltransferase in P . stuartii; Macinga DR et al.; Providencia stuartii contains a chromosomal 2'-N-acetyltransferase {AAC(2')-Ia} involved in the O acetylation of peptidoglycan . The AAC(2')-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed . We report the identification of a locus in P . stuartii, designated aarF, that is required for the expression of AAC(2')-Ia . Northern (RNA) analysis demonstrated that aac(2')-Ia mRNA levels were dramatically decreased in a P . stuartii strain carrying an aarF::Cm disruption . The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone . The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome . An E . coli yigR::Kan mutant was also deficient in ubiquinone content . Complementation studies demonstrated that the aarF and the E . coli yigQR loci were functionally equivalent . The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E . coli chromosome . This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.

J Med Microbiol, 1997 Jun, 46(6), 524 - 7
Media for the detection and recognition of the enteropathogen Providencia alcalifaciens in faeces; Senior BW; A medium (PAM: Providencia alcalifaciens medium) is described that enables the presence of the enteropathogen P . alcalifaciens in faeces to be detected with ease and simplicity . This organism is probably the only oxidase-negative organism likely to be present in tetrathionate broth cultures of faeces that is unable to ferment the mannitol, xylose or galactose present in the medium . Thus the red colonies of P . alcalifaciens appeared quite distinct from the lemon-yellow acid-forming colonies of all the other bacteria that ferment one or more of these sugars . Extensive tests showed the medium to be both highly specific and sensitive in detecting P . alcalifaciens . Two additional media are described that enable the identity of presumptive P . alcalifaciens isolates to be confirmed unequivocally and with ease.

Antimicrob Agents Chemother, 1997 Aug, 41(8), 1749 - 54
An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii; Rather PN et al.; The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase {AAC(2')-Ia} involved in the acetylation of peptidoglycan . In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin . Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase . This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor . AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent . The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above . Biochemical characterization of conditioned media from P . stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable . In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.

J Bacteriol, 1997 Jul, 179(13), 4106 - 14
Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan; Payie KG et al.; The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro . Adaptation of the assay for peptidoglycan N-->O-acetyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gentamicin N-acetyltransferase {EC 2.3.1.59; AAC(2')} . The peptidoglycan from cells of P . stuartii PR50 was prelabelled with 3H by growth in the presence of N-{acetyl-3H}glucosamine . Under these conditions, {3H}acetate was confirmed to be transferred to the C-6 position of peptidoglycan-bound N-acetylmuramyl residues . Isolated cells were subsequently incubated in the presence of various concentrations of gentamicin and tobramycin (0 to 5x MIC) . Analysis of various cellular fractions from isolated cells and spent culture medium by the aminoglycoside-binding phosphocellulose paper assay revealed increasing levels of radioactivity associated with the filters used for whole-cell sonicates of cells treated with gentamicin up to 2 x MIC . Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of cell lysis . Similar results were obtained with tobramycin, but the increasing trend was less obvious . The transfer of radiolabel to either aminoglycoside was not observed with P . stuartii PR100, a strain that is devoid of AAC(2')-Ia . A high-performance anion-exchange chromatography-based method was established to further characterize the AAC(2')-Ia-catalyzed acetylation of aminoglycosides . The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation into two peaks, both of which were collected and confirmed by 1H nuclear magnetic resonance to be the antibiotic . Authentic standards of 2'-N-acetyltobramycin were prepared and were well separated from the parent antibiotic when subjected to the HPLC analysis . By applying this technique, the transfer of radiolabelled acetate from the cell wall polymer peptidoglycan to tobramycin was confirmed . In addition, isolated and purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, and N-acetylglucosamine to tobramycin . These data further support the proposal that AAC(2')-Ia from P . stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides is simply fortuitous.

Biochemistry (Mosc), 1997 May, 62(5), 501 - 8
Structure of the O-specific polysaccharide of the bacterium Providencia alcalifaciens O23 containing a novel component: an amide of D-glucuronic acid with N(epsilon)-(1-carboxyethyl)lysine; Kocharova NA et al.; An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O23 and found to contain D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and N epsilon-(1-carboxyethyl)-N alpha-(D-glucuronoyl)lysine . On the basis of full and partial acid hydrolyses, selective solvolysis with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H, 13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), the following structure of the liner tetrasaccharide repeating unit of the polysaccharide was established {structure: see text}

J Med Microbiol, 1996 Dec, 45(6), 459 - 62
Prevalence of invasive ability and other virulence-associated characteristics in Providencia alcalifaciens strains isolated in São Paulo, Brazil; Guth BE et al.; Providencia alcalifaciens is an invasive enteric pathogen . The present study determined the prevalence of invasive ability in P . alcalifaciens strains isolated in Sao Paulo, Brazil, mainly from patients with diarrhoea . Invasion of HeLa cells was found in 17 (42%) of 41 strains studied . Most (88%) of the invasive strains were isolated from diarrhoeal stools . The invasive property was identified in 50% of P . alcalifaciens strains isolated as pure cultures or from stool samples where no other enteropathogen was identified . All the invasive strains caused actin condensation in infected cells . Plasmid profile analysis showed the presence of plasmids of 35.8-180 kb in 70% of the strains regardless of their invasive ability, suggesting that invasiveness in P . alcalifaciens is not plasmid related . No homology with a probe for gene sequences for invasion of enteroinvasive Escherichia coli and Shigella strains was identified in colony hybridisation assays . The invasive property of P . alcalifaciens was confirmed in the present study, but this characteristic did not predominate among strains isolated from patients with diarrhoea in Sao Paulo City . The presence of other virulence mechanisms and the role of non-invasive P . alcalifaciens strains as a cause of diarrhoea remain to be established.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2350 - 5
Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum; Ainsa JA et al.; A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum . DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da . The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii . This is the second member of a subfamily of AAC(2')-I enzymes to be identified . No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins . The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin . DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M . fortuitum tested . The universal presence of the aac(2')-Ib gene in M . fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.

Tissue Antigens, 1996 Sep, 48(3), 192 - 8
Results of Expedicion Humana . II . Analysis of HLA class II alleles in three African American populations from Colombia using the PCR/SSOP: identification of a novel DQB1*02 (*0203) allele; Trachtenberg EA et al.; PCR/SSOP typing methods were used to analyze the HLA Class II DRB1, DQA1, DQB1 and DPB1 loci of samples from three African American populations of Colombia . Forty samples from the Cauca (Pacific), and twenty samples each from the Choco (North Pacific Coast) and the Providencia (Caribbean island) populations, were collected and the Class II loci analyzed under the auspices of the Expedicion Humana . Despite the limited number of samples analyzed, the African Colombian populations exhibit a very high degree of class II polymorphism . A great diversity of DRB1 alleles was found, with representatives from all serological classes, including 19 DRB1 alleles in the Providencia, 16 in the Cauca and 14 in the Choco groups . In addition, a novel DQB1*02 allele (*0203) was found in two individuals from the Cauca population of the Pacific Coast . The sequence of the DQB1*0203 allele, associated with DR3, differs from DQB1*0201 by only one nucleotide substitution (C-->A) in the second position of codon 57, resulting in an Ala to Asp change . The addition of DQB1*0203 brings the total number of DQB1 alleles identified to date to 26 . HLA class II diversity is much greater in these African Colombian populations than that seen in nearby Amerindian populations . Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.

Antimicrob Agents Chemother, 1996 Sep, 40(9), 2099 - 105
Antimicrobial activity of human pancreatic juice and its interaction with antibiotics; Minelli EB et al.; Pancreatic juice (PJ) should be a factor of variability in the antimicrobial activity of antibiotics eliminated by the pancreas during pancreatic infections . We studied its effects on the activity of antimicrobial drugs with different mechanisms of action . Samples of pure PJ were collected from 16 patients with stabilized external pancreatic fistulas . The antimicrobial activity of the juice at different concentrations (from 1.25 to 100%) alone and in combination with mezlocillin, imipenem, ceftriaxone, gentamicin, ofloxacin, and ciprofloxacin was studied by a microbiological method (continuous turbidimetric recording of bacterial growth) . The human PJ showed dose-dependent antimicrobial activity that increased directly with the concentration . The activity of the antibiotics at bactericidal concentrations were not modified by the PJ, while the combination with subinhibitory concentrations produced the following variable and different effects: (i) additivity with mezlocillin, ceftriaxone, gentamicin, and ciprofloxacin and autonomy (no interaction) with imipenem and ofloxacin against Providencia rettgeri and (ii) additivity with ceftriaxone, ofloxacin, gentamicin, imipenem, and mezlocillin and autonomy with ciprofloxacin against Escherichia coli . In the presence of PJ, fluoroquinolones showed constant positive effects, while beta-lactams showed more variable antimicrobial activity . Antibiotic concentrations and PJ pharmacodynamics are the main factors determining the final effect of the interaction in vitro . These results may be useful in choosing antibiotics for the treatment of pancreatic infections when they are supplemented with the pharmacokinetic data for each drug.

Mol Microbiol, 1996 Feb, 19(3), 511 - 20
aarD, a Providencia stuartii homologue of cydD: role in 2'-N-acetyltransferase expression, cell morphology and growth in the presence of an extracellular factor; Macinga DR et al.; In a search for genes involved in regulation of the 2'-N-acetyltransferase in Providencia stuartii, a mini-Tn5Cm insertion has been isolated in a locus designated aarD . The aarD1::mini-Tn5Cm mutation resulted in a 4.7-fold increase in the levels of beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion and a 32-fold increase in the levels of gentamicin resistance in P . stuartii . The wild-type aarD locus was cloned on a 5.0 kb Cla I fragment and complemented the aarD1 mutation . Nucleotide sequence analysis of this fragment identified two large open reading frames whose deduced products displayed significant amino acid identity, 64% and 64%, respectively, to the CydD and CydC proteins of Escherichia coli, which are involved in formation of the cytochrome d oxidase complex . Physical mapping indicated the aarD1::mini-Tn5Cm insertion was within the open reading homologous to CydD . The strain containing the aarD1 mutation was unable to grow in the presence of toluidine blue or on glycerol minimal media in the presence of zinc, suggesting that aarD is functionally equivalent to cydD . Additional phenotypes resulting from the aarD1 mutation included: altered cell morphology, a reduced growth rate and the inability of cells to grow beyond early log phase . Further examination of this phenomenon revealed that the aarD1 mutant was unable to grow in the presence of a self-produced extracellular factor(s) . This novel phenotype was limited to P . stuartii as E . coli cydD and delta cydAB::kan mutants were also sensitive to a self-produced extracellular factor.

J Bacteriol, 1995 Sep, 177(18), 5350 - 4
Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria; Townsend DE et al.; The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation . The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S . typhimurium corA Mg2+ transport gene . The sequence of the cloned P . stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa . Despite a phenotype similar to that of corA and the close phylogenetic relationship between P . stuartii and S . typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4 . The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products . In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.

J Bacteriol, 1995 Aug, 177(15), 4303 - 10
Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii; Payie KG et al.; A collection of Providencia stuartii mutants which either underexpress or overexpress aac(2')-Ia, the chromosomal gene coding for gentamicin 2'-N-acetyltransferase (EC 2.3.1.59), have been characterized phenotypically as possessing either lower or higher levels of peptidoglycan O acetylation, respectively, than the wild type . These mutants were subjected to both negative-staining and thin-section electron microscopy . P . stuartii PR100, with 42% O acetylation of peptidoglycan compared with 52% O acetylation in the wild type, appeared as irregular rods . In direct contrast, P . stuartii strains PR50.LM3 and PR51, with increased levels of peptidoglycan O acetylation (65 and 63%, respectively), appeared as coccobacilli and chain formers, respectively . Membrane blebbing was also observed with the chain-forming strain PR51 . Thin sectioning of this mutant indicated that it was capable of proper constriction and separation . P . stuartii PM1, when grown to mid-exponential phase, did not have altered peptidoglycan O-acetylation levels, and cellular morphology remained similar to that of wild-type strains . However, continued growth into stationary phase resulted in a 15% increase in peptidoglycan O acetylation concomitant with a change of some cells from a rod-shaped to a coccobacillus-shaped morphology . The fact that these apparent morphological changes were directly related to levels of O acetylation support the view that this modification plays a role in the maintenance of peptidoglycan structure, presumably through the control of autolytic activity.

J Bacteriol, 1995 Jun, 177(12), 3407 - 13
Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii; Macinga DR et al.; The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase {encoded by aac(2')-Ia} in Providencia stuartii . Introduction of aarP into P . stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion . Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation . The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS . The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system . Introduction of aarP on a multicopy plasmid into either P . stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin . Multiple copies of aarP in E . coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E . coli . The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA . Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene . (P.N . Rather, E . Oroz, K.J . Shaw, R . Hare, and G . Miller, J . Bacteriol . 175:6492-6498).

New Microbiol, 1995 Apr, 18(2), 201 - 6
A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene; Burioni R et al.; A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed . The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii . Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E . coli strains carrying pPhoR . Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium . The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation . Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E . coli host.

New Microbiol, 1995 Apr, 18(2), 127 - 33
Engineering human monoclonal antibody fragments: a recombinant enzyme-linked Fab; Burioni R et al.; A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed . pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii . Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase . In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity . Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

J Med Microbiol, 1995 Mar, 42(3), 186 - 90
Characteristics of invasion of HEp-2 cells by Providencia alcalifaciens; Albert MJ et al.; Previous studies with three isolates from diarrhoeal stools suggested that Providencia alcalifaciens is an invasive enteric pathogen that also causes actin condensation in infected cells . These findings were extended in the present study with a further 14 diarrhoeal stool isolates of P . alcalifaciens and HEp-2 cell monolayers for invasion assays . Studies on invasion characteristics with two selected isolates suggested that P . alcalifaciens required prior growth at 37 degrees C for better invasion . Invasion and actin condensation were inhibited by an agent that inhibits microfilament formation, but not by agents that inhibit receptor-mediated endocytosis, microtubule formation, endosome acidification or receptor recycling . In time-course assays with HEp-2 cell monolayers maintained in medium containing gentamicin, P . alcalifaciens showed a small degree of multiplication after invasion of the cells, but viable bacteria could not be recovered over a 24-h period although the integrity of the cell monolayer was preserved during this period.

Protein Sci, 1995 Mar, 4(3), 433 - 41
Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri; Klei HE et al.; Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Bro1) were purified to near homogeneity . The isoforms exhibited comparable enzymatic activities but differed slightly in the molecular weight and pI of their respective alpha-subunit . The origin of this difference was traced to the partial conversion of the N-terminal Gln of the alpha-subunit to pyrrolidonecarboxylic acid (pyro-Glu) . The boundaries of the mature enzyme within the translated DNA sequence of the wild-type propeptide (GenBank M86533) were determined . The results conclusively identified the length of the signal peptide and the position of the spacer cleaved from the propeptide to form the active heterodimer . The molecular weights of the alpha- and beta-subunits, based on these termini, were 23.7 and 62.2 kDa, respectively . Both isoforms were crystallized independently as hexagonal bipyramids up to 0.60 mm in diameter in either space group P6(1)22 or P6(5)22 (a = b = 140.5 A and c = 209.5 A) from ammonium sulfate solutions buffered by 50 mM potassium phosphate at pH 7.5 . The presence of glycerol, although not required, facilitated crystal growth . Native and heavy atom derivative data were collected to 3.0 A resolution, and the calculation of isomorphous replacement phases is under way.

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