The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO. The marRAB operon, transcribed from marO II , controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.Brucellosis is the most common zoonosis in humans. Transmission of this disease occurs mainly by inhalation of infected aerosols, by animal contact, and by conjunctival and gastrointestinal routes. The gastrointestinal route is the most common portal of entry of Brucella in humans through ingestion of raw milk or its products and raw liver or meat (8). Transmission of Brucella melitensis, B. abortus, B. suis, and B. canis in animals also occurs by ingestion of contaminated abortions, discharge materials, or contaminated pasture plants. In contrast, gastrointestinal transmission is not important under natural conditions for B. ovis, where the sexual route seems to be the most probable route of infection (21). Most isolates of B. ovis are urease negative (10).Urease is a multisubunit, nickel-containing enzyme that catalyzes the hydrolysis of urea, yielding ammonia and carbon dioxide. The released ammonia is used by many bacteria as a source of nitrogen, and even for the generation of ATP from a strong ammonia gradient in the case of Ureaplasma urealyticum (38). Moreover, urease is a virulence factor for several human pathogens, and it plays a major role in both urinary and gastrointestinal tract infections, although through different mechanisms (9). In urinary tract infections caused by Proteus mirabilis, urease promotes direct toxicity to renal epithelium cells and kidney stone formation (16,24). In gastrointestinal tract infections, urease allows Helicobacter pylori colonization...

Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was repressed by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. Chromosomally mediated multiple antibiotic resistance (Mar) mutants of Escherichia coli constitutively express the regulated marRAB operon, which is located at 34 min on the E. coli chromosomal map (5, 13). Expression of the operon, consisting of marR, marA, and marB, is normally repressed by MarR but can be induced by diverse compounds such as tetracycline, chloramphenicol, menadione, and salicylates (6,16,28). Mar mutants show increased resistance to multiple antibiotics and structurally unrelated compounds (2,(12)(13)(14). Selected by low concentrations of a single drug, Mar mutants can attain very high levels of resistance if maintained in contact with the selective agent (12). Insertion of Tn5 into marA, the gene for a protein which causes increased antibiotic resistance (11,16,33), completely reversed the resistance phenotype (13).The marRAB operon affects distant chromosomal genes, as revealed by changes seen in proteins separated in two-dimensional gels (14). One effect is to increase expression of micF (8) and thereby indirectly decrease expression of ompF (8). However, the reduced level of OmpF in Mar mutants is only a small component of antibiotic resistance (6, 7), indicating that other genes are involved in the Mar phenotype. To identify the other genes regulated by the marRAB operon and involved in the Mar phenotype, we used a marker fusion approach, with TnphoA (22) and TnlacZ (32). We isolated fusions in which ␤-galactosidase or alkaline phosphatase expression changed when the mar operon was removed. As a result of these studies, we have identified four mar locus-regulated (mlr) genes, three of which have not previously been described. MATERIALS AND METHODSBacterial strains, plasmids, and phages. Bacterial strains and plasmids used in this study are listed in Table 1. The 1.24-kbp BspHI deletion in the mar locus in strain ASS110 was made by homologous recombination following methods described previously (17), with pWY4, a derivative of the temperature-sensitive plasmid pMAK705, bearing the deletion. The presence of the deletion in the recipient strain was verified by Southern blot anal...

The nucleotide sequence of a 3.1-kb region from the chromosome of the Yersinia enterocolitica O:5b strain IP97 containing the gene for an inducible chromosomal cephalosporinase has been determined. The cephalosporinase gene was homologous to other enterobacterial chromosomal cephalosporinase genes, and it was accompanied by a gene homologous to the regulatory ampR gene. The arrangement of genes in the Y. enterocolitica ampCR unit was identical to that in the Enterobacter cloacae and Citrobacter freundii ampCR units.

The nucleotide sequence has been determined of a 1400 bp fragment from the chromosome of Yersinia enterocolitica containing the gene for beta-lactamase I. An ORF of 882 bp was identified, which could code for a polypeptide of 294 amino acids, closely related to other beta-lactamases of molecular class A. Amino acids 1-30 could constitute a signal peptide. The mature protein would be 264 amino acids long with a calculated pI of 6.2. Alignment of the amino acid sequence of the class A beta-lactamases suggested the existence of two subgroups in the same class, and this is discussed in the context of the evolution of the enzymes.