Secretion of the human parathyroid hormone through a microcin type I secretion system in Escherichia coli.

BACKGROUND: Gram negative bacteria possess different secretion systems to export proteins to the extracellular medium. The simplest one, type I secretion system (T1SS), forms a channel across the cell envelope to export proteins in a single step. Peptides secreted by the T1SSs comprise a group of antibiotics, called class II microcins, which carry an amino terminal secretion domain that is processed concomitantly with export. Mature microcins range in size from 60 to 90 amino acids and differ in their sequences. Microcin T1SSs show a high versatility in relation to the peptides they are able to secrete, being mainly limited by the length of the substrates. Different bioactive peptides unrelated to bacteriocins could be secreted by microcin V (MccV) T1SS, while retaining their biological activity. RESULTS: In this work heterologous secretion of two variants of human parathyroid hormone (PTH) by MccV T1SS was evaluated. PTH is a bioactive peptide of 84 amino acids (PTH84), which is involved in the maintenance of bone homeostasis. Currently, a drug corresponding to the active fraction of the hormone, which resides in its first 34 amino acids (PTH34), is commercially produced as a recombinant peptide in Escherichia coli. However, research continues to improve this recombinant production. Here, gene fusions encoding hybrid peptides composed of the MccV secretion domain attached to each hormone variant were constructed and expressed in the presence of microcin T1SS in E. coli cells. Both PTH peptides (PTH34 and PTH84) were recovered from the culture supernatants and could be confirmed to lack the MccV secretion domain, i.e. microcin T1SS efficiently recognised, processed and secreted both PTH variants. Furthermore, the secreted peptides were stable in the extracellular medium unlike their unprocessed counterparts present in the intracellular space. CONCLUSION: The successful secretion of PTH variants using MccV T1SS could be considered as a new alternative for their production, since they would be recovered directly from the extracellular space without additional sequences. Furthermore, it would be a new example revealing the potential of microcin type I secretion systems to be conceived as a novel strategy for the production of recombinant peptides in E. coli.

Characterization of Streptococcus equi subsp. zooepidemicus isolates containing lnuB gene responsible for the L phenotype.

Within the framework of the ß-hemolytic streptococci surveillance carried out by the National Reference Laboratory from Uruguay, three putative Streptococcus equi subsp. zooepidemicus (SEZ) were received from different health centers. Being these the first reports associated with human infections in Uruguay, the objective of this work was to confirm their identification, to determine their genetic relationship and to study their antibiotic susceptibility. Using four different methods, they were identified as SEZ, a subspecies which has been described as the etiologic agent of rare and severe zoonosis in a few cases in other countries. The three isolates presented different pulsotypes by PFGE; however, two of them appeared to be related and were confirmed as ST431 by MLST, while the remaining isolate displayed ST72. Their resistance profile exhibited an unexpected feature: despite all of them were susceptible to macrolides, they showed different levels of resistance to clindamycin, i.e. they had the so-called "L phenotype". This rare trait is known to be due to a nucleotidyl-transferase, encoded by genes of the lnu family. Although this phenotype was previously described in a few SEZ isolates, its genetic basis has not been studied yet. This was now analyzed by PCR in the three isolates and they were found to contain a lnuB gene. The lnuB sequence was identical among the three isolates and with many lnuB sequences deposited in data banks. In conclusion, for the first time in Uruguay, three SEZ isolates recovered from non-epidemiologically related cases of human invasive infection were identified. Moreover, this is the first report about the presence of a lnu gene in the S. equi species, revealing the active lateral spread of the lnuB in a new streptococcal host.

Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12.

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

Microcin H47 system: an Escherichia coli small genomic island with novel features.

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.

Microcins and urovirulence in Escherichia coli.

Urinary tract infections are among the most common infectious diseases encountered in humans and Escherichia coli is their leading etiologic agent. Uropathogenic E. coli encompasses a group of bacteria possessing a variable virulence gene assortment. It is generally agreed that many urovirulence factors remain to be discovered and that this information is required to gain knowledge on the pathogenic processes underlying the different clinical presentations of urinary tract infections. The production of higher-molecular-mass microcins, a group of ribosomally-synthesized peptide antibiotics comprising microcins H47, I47, E492, M and ColV, has been proposed as a virulence trait of some uropathogenic E. coli. To study this possibility, clones producing any of these microcins were selected from a collection of 160 Gram-negative clinical isolates from urine cultures and their virulence profile was analyzed. The study consisted in surveying genetic loci known to be relevant to urinary tract infection caused by E. coli. Depending on the type of microcin produced, different virulence patterns were observed which seemed to be determined by the degree of compatibility between virulence and microcin loci. In conclusion, results pointed to a relationship between higher-molecular-mass microcins and urovirulence.

Modular structure of microcin H47 and colicin V.

Microcins are gene-encoded peptide antibiotics produced by enterobacteria that act on strains of gram-negative bacteria. In this work, we concentrated on higher-molecular-mass microcins, i.e., those possessing 60 or more amino acids. They can be subdivided into unmodified and posttranslationally modified peptides. In both cases, they exhibit conserved C-terminal sequences that appear to be characteristic of each subgroup. In the hypothesis that these sequences could correspond to domains, gene fusions between the activity genes for the unmodified microcin colicin V and the modified microcin H47 were constructed. These two microcins differ in their mode of synthesis, uptake, target, and specific immunity. Through this experimental approach, chimeric peptides with exchanged C-terminal sequences were encoded. Cells carrying the fusions in different genetic contexts were then assayed for antibiotic production. Many of them produced antibiotic activities with recombinant properties: the toxicity of one microcin and the mode of uptake of the other. The results led to the identification of a modular structure of colicin V and microcin H47, with the recognition of two domains in their peptide chains: a toxic N-terminal domain and an uptake C-terminal domain. This modular design would be shared by other microcins from each subgroup.

Comparative analysis of chromosome-encoded microcins.

Microcins are ribosomally synthesized peptide antibiotics that are produced by enterobacterial strains. Although the first studies concentrated on plasmid-encoded activities, in the last years three chromosome-encoded microcins have been described: H47, E492, and M. Here, a new microcin, I47, is presented as a fourth member of this group. Common features exhibited by chromosome-encoded microcins were searched for. The comparison of the genetic clusters responsible for microcin production revealed a preserved general scheme. The clusters essentially comprise a pair of activity-immunity genes which determine antibiotic specificity and a set of microcin maturation and secretion genes which are invariably present and whose protein products are highly homologous among the different producing strains. A strict functional relationship between the maturation and secretion pathways of microcins H47, I47, and E492 was demonstrated through genetic analyses, which included heterologous complementation assays. The peptide precursors of these microcins share a maturation process which implies the addition of a catecholate siderophore of the salmochelin type. Microcins thus acquire the ability to enter gram-negative cells through the catechol receptors. In addition, they employ a common mode of secretion to reach the external milieu by means of a type I export apparatus. The results presented herein lead us to propose that chromosome-encoded microcins constitute a defined subgroup of peptide antibiotics which are strictly related by their modes of synthesis, secretion, and uptake.

Involvement of enterobactin synthesis pathway in production of microcin H47.

Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an Escherichia coli clinical isolate which is active on strains of gram-negative bacteria. Its uptake by E. coli K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism. The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated. Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores: mchA and mchS1 are iroB and iroD homologues, respectively, and mchS4 was found to promote the production of the catecholate siderophore enterobactin. The possible relationship between enterobactin synthesis and MccH47 production was studied. Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the ent genetic cluster, the production of both the siderophore and MccH47 was restored. Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis. The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.