In vivo dynamics of type 1 fimbria regulation in uropathogenic Escherichia coli during experimental urinary tract infection.

Escherichia coli is the primary cause of uncomplicated infections of the urinary tract including cystitis. More serious infections, characterized as acute pyelonephritis, can also develop. Type 1 fimbriae of E. coli contribute to virulence in the urinary tract; however, only recently has the expression of the type 1 fimbriae been investigated in vivo using molecular techniques. Transcription of type 1 fimbrial genes is controlled by a promoter that resides on a 314-bp invertible element capable of two orientations. One places the promoter in the ON orientation, allowing for transcription; the other places the promoter in the OFF orientation, preventing transcription. A PCR-based assay was developed to measure the orientation of the invertible element during an experimental urinary tract infection in mice. Using this assay, it was found that the percentage of the population ON in urine samples correlated with the respective CFU per gram of bladder (P = 0.0006) but not with CFU per gram of kidney (P > 0.069). Cystitis isolates present in the urine of mice during the course of infection had a higher percentage of their invertible elements in the ON orientation than did pyelonephritis isolates (85 and 34%, respectively, at 24 h; P < 0.0001). In general, cystitis isolates, unlike pyelonephritis isolates, were more likely to maintain their invertible elements in the ON orientation for the entire period of infection. E. coli cells expressing type 1 fimbriae, expelled in urine, were shown by scanning electron microscopy to be densely packed on the surface of uroepithelial cells. These results suggest that expression of type 1 fimbriae is more critical for cystitis strains than for pyelonephritis strains in the early stages of an infection during bladder colonization.

In vivo phase variation of Escherichia coli type 1 fimbrial genes in women with urinary tract infection.

Type 1 fimbriae, expressed by most Escherichia coli strains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by SnaBI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First, E. coli strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with E. coli CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.

spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis.

The spo0J gene of Bacillus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J). These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A. In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and Spo0J are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2, spo0J was found to be required for normal chromosome partitioning as well as for sporulation. spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.