The colicin G producer Escherichia coli CA46, the colicin H producer E. coli CA58 and E. coli Nissle 1917 (DSM 6601) were shown to produce microcin H47 and the newly described microcin M. Both microcins were exported like colicin V by an RND-type export system, including TolC. The gene cluster encoding microcins H47 and M in strains CA46 and CA58 is nearly identical to that in strain DSM 6601, except that two additional genes are included. A Fur box identified in front of the microcin-encoding genes explained the observed iron regulation of microcin production. The catecholate siderophore receptors Fiu, Cir and FepA from E. coli and IroN, Cir and FepA from Salmonella were identified as receptors for microcins M, H47 and E492. IroN takes up the glucose-containing catecholate siderophore salmochelin, whose synthesis is encoded in the iro gene cluster found in Salmonella and certain, often uropathogenic, E. coli strains. A gene in this iro cluster, iroB, which encodes a putative glycosyltransferase, was also found in the microcin H47/M and microcin E492 gene clusters. These microcins could aid the producing strain in competing against enterobacteria that utilize catecholate siderophores.

Microcins are low-molecular-weight, ribosomally produced, highly stable, bacterial-inhibitory molecules involved in competitive, and amensalistic interactions between Enterobacteriaceae in the intestine. These interactions take place in a highly complex chemical landscape, the intestinal eco-active chemosphere, composed of chemical substances that positively or negatively influence bacterial growth, including those originated from nutrient uptake, and those produced by the action of the human or animal host and the intestinal microbiome. The contribution of bacteria results from their effect on the host generated molecules, on food and digested food, and organic substances from microbial origin, including from bacterial degradation. Here, we comprehensively review the main chemical substances present in the human intestinal chemosphere, particularly of those having inhibitory effects on microorganisms. With this background, and focusing on Enterobacteriaceae, the most relevant human pathogens from the intestinal microbiota, the microcin’s history and classification, mechanisms of action, and mechanisms involved in microcin’s immunity (in microcin producers) and resistance (non-producers) are reviewed. Products from the chemosphere likely modulate the ecological effects of microcin activity. Several cross-resistance mechanisms are shared by microcins, colicins, bacteriophages, and some conventional antibiotics, which are expected to produce cross-effects. Double-microcin-producing strains (such as microcins MccM and MccH47) have been successfully used for decades in the control of pathogenic gut organisms. Microcins are associated with successful gut colonization, facilitating translocation and invasion, leading to bacteremia, and urinary tract infections. In fact, Escherichia coli strains from the more invasive phylogroups (e.g., B2) are frequently microcinogenic. A publicly accessible APD3 database http://aps.unmc.edu/AP/ shows particular genes encoding microcins in 34.1% of E. coli strains (mostly MccV, MccM, MccH47, and MccI47), and much less in Shigella and Salmonella (<2%). Some 4.65% of Klebsiella pneumoniae are microcinogenic (mostly with MccE492), and even less in Enterobacter or Citrobacter (mostly MccS). The high frequency and variety of microcins in some Enterobacteriaceae indicate key ecological functions, a notion supported by their dominance in the intestinal microbiota of biosynthetic gene clusters involved in the synthesis of post-translationally modified peptide microcins.

Polymorphisms in the rifampin resistance mutation frequency (f) were studied in 696 Escherichia coli strains from Spain, Sweden, and Denmark. Of the 696 strains, 23% were weakly hypermutable (4 ؋ 10 ؊8 < f < 4 ؋ 10 ؊7 ), and 0.7% were strongly hypermutable (f > 4 ؋ 10 ؊7 ). Weak mutators were apparently more frequent in southern Europe and in blood isolates (38%) than in urinary tract isolates (25%) and feces of healthy volunteers (11%).Microbial evolution is dependent on two opposing forces, the maintenance of genetic information and the generation of some suitable level of genetic variation on which selection can act. In most cases, genetic variation is assured by errors in DNA replication, determined by the accuracy of DNA polymerases and various DNA repair systems. Particular environmental characteristics will influence selection of the optimal amount of genetic variation for a given organism with a specific population structure. If the environment changes rapidly in time or is heterogeneous, variants with increased mutation rates will tend to be selected, since they have an increased probability of forming beneficial mutations. Conversely, if the environment is constant, as the organism becomes maximally adapted, mutation rates tend to decrease because of the costs associated with deleterious mutations (4, 6, 7). These considerations suggest that environment-dependent polymorphisms in mutation frequency can be expected in nature.Mutation frequencies were determined in a collection of 696 Escherichia coli strains obtained from 2000 to 2003. Of the 696 E. coli strains, 300 were from Spain (100 from positive urine cultures, 100 from blood cultures, and 100 from the stools of young healthy volunteers), 170 were from Denmark (blood cultures), and 226 were from Sweden (urinary tract cultures from outpatients). Each Luria-Bertani (LB) tube was inoculated with an independent colony obtained from a blood agar plate; three LB tubes were used. After 24 h of incubation, appropriate dilutions were seeded onto LB agar plates and LB agar plates containing rifampin (100 g/ml), and colony counts were performed after 24 or 48 h, respectively. Mutation frequencies are reported as a proportion of the number of rifampin-resistant colonies to the total viable count. The results corresponded to the mean value obtained in three independent experiments that were repeated in cases of suspected jackpots.Categories were established considering the distribution of frequencies of the 696 E. coli strains (Fig. 1). A strain was considered normomutable when the mutation frequency (f) was at or close to the modal point of the distribution of mutation frequencies; for practical purposes, it was established as 8 ϫ 10 Ϫ9 Ͻ f Ͻ 4 ϫ 10 Ϫ8 . Strains were considered weak mutators if their frequency was 4 ϫ 10 Ϫ8 Յ f Ͻ 4 ϫ 10 Ϫ7 and strong mutators if f Ն 4 ϫ 10 Ϫ7 . Hypomutable strains were defined as strains with f Յ 8 ϫ 10 Ϫ9 . A sharp peak in the frequency distribution was always found at 10 Ϫ8 . From this value, a few strains had lower mutatio...

A clinical Escherichia coli strain highly resistant to the combinations of amoxicillin-clavulanate, ampicillinsulbactam, and piperacillin-tazobactam was isolated from a patient with a community-acquired urinary tract infection who was previously treated with amoxicillin-clavulanate. These resistances were carried by a 45-kb conjugative plasmid encoding for a single 1-lactamase with a pI of 5.4. Cloning and sequencing of the new 13-lactamase, revealed identity with the blaT) gene encoding the TEM-1 1-lactamase except for a replacement of the methionine residue at position 67 by isoleucine and of the methionine residue at position 180 by threonine. Both mutations were segregated by the construction of hybrid genes, and only the mutation at methionine at position 67 was related to resistance to the suicide inhibitors. The inhibitory effects of clavulanate, sulbactam, and tazobactam on the TEM-1 enzyme were substantially decreased in comparison with those on IRT-3, as indicated by the 50%o inhibitory concentrations.Bacterial resistance to penicillins and cephalosporins represents an increasing problem in the chemotherapy of gramnegative infections. The emergence and worldwide spread of

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [ 3 H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.Penicillin-binding proteins (PBPs) are cytoplasmic membrane enzymes involved in the last steps of peptidoglycan biosynthesis (29). They are able to bind ␤-lactams covalently at a conserved active serine residue because of their structural homology with the natural substrate (D-Ala-D-Ala) for transpeptidation (23, 42). Up to 10 PBPs have been identified in Escherichia coli by using different labelled ␤-lactam antibiotics and different electrophoretic conditions (PBP1a,42). PBP1c was seen only by using a 125 I-Bolton-Hunter derivative of ampicillin (38), and PBP8 has been shown to be an OmpT-mediated proteolytic degradation product of PBP7 (22). The genes coding for the eight classical PBPs (ponA, ponB, pbpA, pbpB, dacB, dacA, dacC, and pbpG, respectively) have previously been cloned and sequenced.The high-molecular-mass PBPs (PBP1a, -1b, -2, and -3) are believed to be dual transpeptidase-transglycosylases. They catalyze glycan chain elongation and peptidoglycan cross-linkings (17). These enzymes are essential for cell growth and survival, since they are the primary targets of ␤-lactam antibiotic action (44,46). Their inactivation by antibiotics or by mutation results in cell death. PBP2 is essential for the maintenance of the rod shape of cells during elongation (44), whereas PBP3 is essential for the formation of the septum during cell division (41).The low-molecular-mass PBPs (PBP4, -5, -6, and -7) are dispensable, as their inactivation by mutation does not affect bacterial growth and division. PBP7 is a DD-endopeptidase (39); PBP4 is essentially a DD-endopeptidase, but it has also a weak DD-carboxypeptidase activity (26). PBP5 and PBP6, which account for 80% of the penicillin-binding capacity of cells, are the major DD-carboxypeptidases (42). The physiological role of the low-molecular-mass PBPs remains to be elucidated.Here we present dacD, an E. coli gene which encodes a novel PBP with a molecular mass equivalent to those of PBP5 and PBP6. Like those proteins, the novel PBP, PBP6b, has DDcarboxypeptidase activity. We have also identified the gene homo...