A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.Urinary tract infections (UTIs) are a serious health concern. Forty to 50% of women experience at least one UTI, leading to an estimated 8 million annual physician visits in the United States alone (39, 46). Uropathogenic Escherichia coli (UPEC) is by far the most common etiological agent of all UTIs. UPEC strain CFT073, derived from the clonal group O6:K2:H1 (26), was originally isolated from the blood and urine of a woman diagnosed with acute pyelonephritis (28). It is considered a prototype of the O6 serogroup, one of the most prevalent UPEC clonal lines (23,24). The virulence of this strain was reproduced in the well-established CBA mouse model of ascending UTI (28). In addition to numerous virulence studies, the genome of E. coli CFT073 has recently been sequenced and compared to that of enterohemorrhagic E. coli EDL933 and the nonpathogenic laboratory strain E. coli MG1655 (42).Mutations have been introduced into a number of candidate virulence genes in UPEC, leading to attenuated mutants in experimental UTI. These include fim, encoding type 1 fimbria (7, 13), sat, encoding secreted autotransporter toxin (14), cnf-1, encoding cytotoxic necrotizing factor (36), tonB, involved in iron transport (40), proP, involved in osmoprotectant transport (8), and degS (35). Large-scale screens for virulence factors of UPEC have also identified factors that aid UPEC during growth in urine (38) and have implicated capsule, lipopolysaccharide, ...

Uropathogenic Escherichia coli is the most common etiological agent of urinary tract infections. Bacteria can often express multiple adhesins during infection in order to favor attachment to specific niches within the urinary tract. We have recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during urinary tract infection. Here, we examine whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli strain CFT073, which harbors genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 h. No differences in the colonization of these mutants were observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant that was incapable of expressing either type 1 or P fimbriae compensated by synthesizing F1C fimbriae.Uropathogenic Escherichia coli (UPEC) strains cause the majority of all urinary tract infections (UTIs). Forty to 50% of women experience at least one UTI during their lifetime, leading to an estimated 8 million physician visits annually in the United States (39, 55). Recent efforts to understand the mechanisms of virulence in this important pathogen include the sequencing of UPEC (52), complete transcriptome analysis (45), signature-tagged mutagenesis (4), and differential fluorescence induction (33). These studies collectively implicate adhesins, iron acquisition systems, capsules, lipopolysaccharides, and toxins in UPEC pathogenesis.Adherence to host tissues is often the first step towards colonization; thus, adhesins are essential for pathogenesis. The recent sequencing of UPEC strain CFT073, along with previous virulence studies, has predicted or demonstrated as many as 12 fimbrial gene clusters in this strain (5, 17, 52). Many fimbrial and afimbrial adhesins are phase variable (28, 34), including the most ubiquitous type 1 fimbriae encoded by the fim gene cluster. The expression of type 1 fimbriae is controlled by a promoter situated on an invertible element of DNA, also referred to as the fim switch (1). Bacteria are phase on, and type 1 fimbriae are expressed when the promoter faces the direction of fimA, which encodes the main structur...

The arginine deiminase (AD) system (ADS) is one of two major ammonia-generating pathways in the oral cavity that play important roles in oral biofilm pH homeostasis and oral biofilm ecology. To initiate a study of the Streptococcus gordonii ADS, the ADS gene cluster was isolated from subgenomic DNA libraries of S. gordonii DL1 by using an arcB-specific probe. Nucleotide sequence analysis revealed six open reading frames (ORFs) that were arranged contiguously; the first five ORFs were transcribed in the same direction, as an apparent operon, and the sixth was transcribed in the opposite direction. The ORFs were found to share significant homologies and to correspond closely in molecular mass to previously characterized arc genes; thus, they were designated arcA (AD), arcB (ornithine carbamyltransferase), arcC (carbamate kinase), arcD (arginine-ornithine antiporter), arcT (dipeptidase), and arcR (regulator). A putative 70 promoter (ParcA [TTGTGT-N 19 -T AGAAT]) was mapped 5 to arcA by primer extension, and the expression of ParcA was shown to be inducible by arginine and repressible by glucose, in agreement with AD specific activities measured in the wild-type strain. To investigate the function of ArcR in the differential expression of the arc operon, arcR was insertionally inactivated by a KM resistance marker flanked by T4 transcription/translation termination signals, and the expression of ParcA was monitored by primer extension in the wild-type and ArcR-deficient strains. Lower levels of arcA expression, as well as lower levels of AD activity, were consistently observed in the ArcR-deficient strain compared to wild-type cells, regardless of the growth conditions. Thus, ArcR is a transcriptional activator that is required for induction and optimal expression of the S. gordonii ADS gene cluster.The arginine deiminase (AD) system (ADS) is a three-enzyme pathway that catalyzes the conversion of arginine to ornithine, ammonia, and CO 2 with the concomitant production of ATP (16). Arginine is first hydrolyzed by AD, encoded by arcA, to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamylphosphate via a catabolic ornithine carbamyltransferase (cOTC) encoded by arcB. Finally, carbamate kinase (CK), encoded by arcC, transfers phosphate from carbamylphosphate to ADP to produce ATP, CO 2 , and ammonia. In some cases, an arginine-ornithine antiporter (ArcD), which catalyzes the uptake of arginine and concomitant export of ornithine, and a putative transaminase or peptidase (ArcT) have been found to be part of arc gene clusters (37).Arginine metabolism via the ADS is widely distributed in both bacteria and archaebacteria, and the primary structures of the enzymes involved in the pathway have been reasonably conserved throughout evolution. In contrast, the physiologic role and genetic regulation of expression of the ADS vary among microorganisms. For instance, both Bacillus licheniformis (32) and Pseudomonas aeruginosa (26) utilize the ADS exclusively under anaerobic conditions and the pr...

Type 1 fimbria is a proven virulence factor of uropathogenic Escherichia coli (UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as "on," and the opposite orientation, in which no transcription occurs, is defined as "off." During the course of a urinary tract infection, we have observed that the infecting E. coli population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (P < 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (P < 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (P ‫؍‬ 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder.

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