Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.

DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.

Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene.

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

Urethral obstruction of 6 hours or less causes bacteriuria, bacteremia, and pyelonephritis in mice challenged with "nonuropathogenic" Escherichia coli.

Urethral obstruction may be caused by prostatic hypertrophy, urethral stricture, or encrustation of a urethral-catheter lumen. Bacteriuria often complicates these obstructions. The sequelae include fever, acute pyelonephritis, chronic renal inflammation, and death. We hypothesized that even brief obstruction of the urinary tract containing a nonvirulent bacterium would result in these complications. Mice challenged transurethrally with Escherichia coli FN414, which is rapidly eliminated from normal mice without causing bacteriuria, bacteremia, or renal pathology, were subjected to reversible urethral obstruction by coating the urethral meatus with collodion for 1, 3, or 6 h. The majority of mice obstructed for 1 h demonstrated parenchymal renal inflammation 48 h later. At the end of 3 h of obstruction, 9 of 10 mice were bacteremic; some bacteremias were present at 48 h after removal of the obstruction. At that time, more severe renal inflammation was seen in these mice. As little as 6 h of obstruction resulted not only in the acute changes described above but also in chronic renal inflammation and fibrosis in the majority of animals sacrificed 3 and 6 weeks later. Additional studies demonstrated that urethral obstruction enhanced the uropathogenicity of another nonpathogenic E. coli strain (K-12 strain HB101) and caused more severe renal lesions in mice challenged with E. coli CFT073, isolated from a patient with symptoms of pyelonephritis. These findings demonstrate that brief urethral obstruction may (i) induce organisms which are cleared rapidly from the normal urinary tract to cause bacteriuria, bacteremia, and pyelonephritis and (ii) intensify the renal lesions caused by a uropathogen.

Early colonic damage and invasion of Campylobacter jejuni in experimentally challenged infant Macaca mulatta.

Experimental challenge studies with Campylobacter jejuni were conducted in 3.5-month-old infant Macaca mulatta. One infant monkey (92-1) was challenged with 2.7 x 10(10) cfu of strain 78-37. A second infant was infected intentionally by natural transmission. The infants developed diarrhea 32 h after challenge of infant 92-1. Electron microscopic observations indicate that cell invasion is the primary mechanism of colon damage and diarrheal disease caused by C. jejuni. Intracellular C. jejuni were located in membrane-bound vacuoles and were free in the cytoplasm. Damaged epithelial cells exhibited premature apoptosis and were exfoliated into the lumen of the colon. C. jejuni were also located extracellularly in the mucosa and submucosa. Some cells had dilated endoplasmic reticulum, indicating possible alteration in ion and water transport.

Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a significant cause of bacteriuria and acute pyelonephritis in humans, produces urease. This high-molecular-weight, multimeric, cytoplasmic enzyme hydrolyzes urea to ammonia and carbon dioxide. To assess the role of urease in colonization, urolithiasis, and acute pyelonephritis in an animal model of ascending urinary tract infection, we compared a uropathogenic strain of P. mirabilis with its isogenic urease-negative mutant, containing an insertion mutation within ureC, the gene encoding the large subunit of the enzyme. Mice challenged transurethrally with the parent strain developed significant bacteriuria and urinary stones. The urease-negative mutant had a 50% infective dose of 2.7 x 10(9) CFU, a value more than 1,000-fold greater than that of the parent strain (2.2 x 10(6) CFU). The urease-positive parent strain reached significantly higher concentrations and persisted significantly longer in the bladder and kidney than did the mutant. Indeed, in the kidney, the parent strain increased in concentration while the mutant concentration fell so that, by 1 week, the parent strain concentration was 10(6) times that of the mutant. Similarly, the urease-positive parent produced significantly more severe renal pathology than the mutant. The initial abnormalities were in and around the pelvis and consisted of acute inflammation and epithelial necrosis. By 1 week, pyelitis was more severe, crystals were seen in the pelvis, and acute pyelonephritis, with acute interstitial inflammation, tubular epithelial cell necrosis, and in some cases abscesses, had developed. By 2 weeks, more animals had renal abscesses and radial bands of fibrosis. We conclude that the urease of P. mirabilis is a critical virulence determinant for colonization, urolithiasis, and severe acute pyelonephritis.

The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans. Western blot analysis of the recombinant phosphotriesterase produced by S. lividans demonstrated only the mature form extracellularly but both processed and unprocessed forms in cell-associated samples. To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces beta-galactosidase signal sequence for the Flavobacterium signal sequence. This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated. These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins.

Investigation of an outbreak of bloody diarrhea: association with endoscopic cleaning solution and demonstration of lesions in an animal model.

UNLABELLED: OUTBREAK INVESTIGATION: An outbreak of diarrhea, bloody diarrhea, and abdominal cramps occurred among persons undergoing flexible sigmoidoscopy at a branch clinic of a local health center. Illness was associated with use of sigmoidoscopes cleaned by one clinic assistant and appeared to be caused by 2% glutaraldehyde disinfectant solution left in the instruments after cleaning. ANIMAL STUDIES: In subsequent animal studies, colonic instillation of 2% glutaraldehyde solutions caused bloody diarrhea and a distinctive pattern of mucosal damage; similar changes were seen in a review of pathologic samples from other human cases of glutaraldehyde disinfectant-associated diarrhea. CONCLUSION: Our data indicate that improper endoscopic reprocessing can result in serious illness and underscore the importance of adequate training and quality control in this area.

Cold agglutinins in cats with haemobartonellosis.

Coombs-positive anemia developed in cats inoculated with Haemobartonella felis. Cold agglutinins were detected in serum during the acute stage of the disease when anemia was present. The cold agglutinating activity was associated with IgM, was demonstrated at 4 C, and was abolished by treatment of sera with 2-mercaptoethanol. At 4 C, the sera from infected cats agglutinated or lysed parasitized autologous erythrocytes or normal erythrocytes pretreated with neuraminidase. These data indicate that cold agglutinins are associated with haemobartonellosis and suggest that immunologic responses to erythrocytic antigens have a role in the anemia.

Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.

S-adenosylhomocysteine metabolism in Streptomyces flocculus.

S-Adenosylhomocysteine metabolism was studied in cell extracts of streptonigrin-producing Streptomyces flocculus. The major route of metabolism was found to be deamination to form S-inosylhomocysteine. The metabolite was purified by high-performance liquid chromatography and identified by its UV and nuclear magnetic resonance spectra and by its chemical degradation to hypoxanthine.