Publications

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABD(II) operon of the UPEC isolate J96, encoding the toxin alpha-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the alpha-hemolysin encoded in hlyCABD(II) by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the alpha-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABD(II) expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABD(II) is promoted at 37 degrees C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABD(II) operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.

JTD Keywords: gene regulation, ppgpp, upec, Alpha-hemolysin, Bacterial signal molecule, Determinants, Environmental-regulation, Gene regulation, H-ns, Ppgpp, Protein, Regulator, Rfah, Secretion, Transcription, Upec, Virulence, Α-hemolysin

Background: Gene duplication underlies a significant proportion of gene functional diversity and genome complexity in both eukaryotes and prokaryotes. Although several reports in the literature described the duplication of specific genes in E. coli, a detailed analysis of the extent of gene duplications in this microorganism is needed. Results: The genomes of the E. coli enteroaggregative strain 042 and other pathogenic strains contain duplications of the gene that codes for the global regulator Hha. To determine whether the presence of additional copies of the hha gene correlates with the presence of other genes, we performed a comparative genomic analysis between E. coli strains with and without hha duplications. The results showed that strains harboring additional copies of the hha gene also encode the yeeR irmA (aec69) gene cluster, which, in turn, is also duplicated in strain 042 and several other strains. The identification of these duplications prompted us to obtain a global map of gene duplications, first in strain 042 and later in other E. coli genomes. Duplications in the genomes of the enteroaggregative strain 042, the uropathogenic strain CFT073 and the enterohemorrhagic strain O145:H28 have been identified by a BLASTp protein similarity search. This algorithm was also used to evaluate the distribution of the identified duplicates among the genomes of a set of 28 representative E. coli strains. Despite the high genomic diversity of E. coli strains, we identified several duplicates in the genomes of almost all studied pathogenic strains. Most duplicated genes have no known function. Transcriptomic analysis also showed that most of these duplications are regulated by the H-NS/Hha proteins. Conclusions: Several duplicated genes are widely distributed among pathogenic E. coli strains. In addition, some duplicated genes are present only in specific pathotypes, and others are strain specific. This gene duplication analysis shows novel relationships between E. coli pathotypes and suggests that newly identified genes that are duplicated in a high percentage of pathogenic E. coli isolates may play a role in virulence. Our study also shows a relationship between the duplication of genes encoding regulators and genes encoding their targets.

JTD Keywords: Escherichia coli 042, Gene duplication, H-NS, Hha, Pathotypes

The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer. The nucleoid-associated protein H-NS silences unwanted expression of acquired foreign DNA. Ali and colleagues recently identified which horizontally-acquired genes are targeted by H-NS in Salmonella to avoid fitness loss. The reported data strengthen our view about the role of H-NS in bacterial evolution driven by horizontal gene transfer.

JTD Keywords: HGT, H-NS, StpA, Salmonella, SPI1

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence.

JTD Keywords: Salmonella, Gene regulation, Motility, Pathogenicity island, H-NS, HHA, STPA

Background The HilA protein is the master regulator of the Salmonella pathogenicity island 1 (SPI1). EilA and YgeH proteins show a moderate similarity to HilA and are encoded in pathogenicity islands from several E. coli strains, both pathogenic and non-pathogenic. In the present work we characterize the YgeH protein from the enteroaggregative E. coli strain 042 (locus tag EC042_3050). Results We show that both E. coli 042 YgeH and EilA proteins are able to functionally replace HilA in Salmonella. Interestingly, this is not the rule for all YgeH proteins: the YgeH protein from the enterohaemorragic E. coli strain O157 appears to be non-functional. ygeH expression is not influenced by growth osmolarity or temperature, and moderately increases in cells entering the stationary phase. H-NS represses ygeH expression under all growth conditions tested, and binds with specificity to the ygeH promoter region. As expected, expression of ETT2 (Escherichia coli type 3 secretion system 2) genes requires YgeH: ETT2 operons are downregulated in a ygeH mutant. Accordingly, since H-NS represses ygeH expression, ETT2 expression is significantly increased in an hns mutant. Conclusion E. coli 042 YgeH protein is functional and able to replace HilA in Salmonella. ETT2 gene expression requires YgeH activity which, in turn, is subjected to H-NS silencing.

JTD Keywords: HilA, YgeH, E. coli 042, H-NS

H-NS and Hha belong to the nucleoid-associated family of proteins and modulate gene expression in response to environmental stimuli. Genes coding for these proteins can be either chromosomally or plasmid-encoded. In this work, we analyse the regulatory role of the Hha protein encoded in the virulence plasmid of the enterohemorrhagic Escherichia coli O157:H7 (HhapO157). This plasmid is present in all clinical isolates of E. coli O157:H7 and contributes to virulence. Both, HhapO157 and E. coli O157:H7-chromosomal Hha (Hhachr) exhibit a significant degree of similarity. The hha gene from plasmid pO157 is transcribed from its own putative promoter and is overexpressed in a chromosomal hha mutant. As its chromosomal counterpart, HhapO157 is able to interact with H-NS. Remarkably, HhapO157 targets only a subset of the genes modulated by Hhachr. This has been evidenced by both assaying the ability of HhapO157 to complement expression of a specific operon (i.e., the haemolysin operon) and by comparing the global transcriptome of the wt strain and its hhap, hhac and hhapc mutant derivatives. HhapO157 and Hhachr share some common regulatory features, however they also display specific targeting of some genes and even a different modulatory role in some others.

JTD Keywords: E. coli O157:H7, Hha, H-NS, Plasmid, pO157, Nucleoid-associated proteins

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin a-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin a-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly(-)). Generation of Hly(-) clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly(-) clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly(-) derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly(-) clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

JTD Keywords: Promotes horizontal dissemination, Enterica serovar typhimurium, Escherichia-coli strains, Insertion-sequence IS91, H-NS, Adaptive amplification, Pathogenicity islands, Hemolysin

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of horizontally acquired genes in Enterobacteria. Systematic mutations of conserved acidic residues in Hha have allowed the identification of D48 as an essential residue for H-NS binding and the involvement of E25. Mutations of these residues resulted in deregulation of sensitive genes in vivo. D48 is only partially solvent accessible, yet it defines the functional binding interface between Hha and H-NS confirming that Hha has to undergo a conformational change to bind H-NS. Exposed acidic residues, such as E25, may electrostatically facilitate and direct the approach of Hha to the positively charged region of H-NS enabling the formation of the final complex when D48 becomes accessible by a conformational change of Hha. Structured summary of protein interactions: YdgT and H-NS bind by nuclear magnetic resonance (View interaction) Hha and H-NS bind by nuclear magnetic resonance (View Interaction 1, 2, 3) Hha physically interacts with H-NS by pull down (View Interaction 1, 2).

JTD Keywords: Nucleoid associated protein, H-NS, Hha, Transcription repression

The nucleoid-associated protein H-NS can be either chromosomal- or plasmid-encoded. We provide in this report evidence indicating that chromosomal- and plasmid-encoded H-NS proteins may differ in their functional properties. The modulatory function of chromosomal H-NS is antagonized by the H-NSTEPEC protein. We show that the H-NS protein encoded by the IncHI plasmid R27 (H-NSR27) is less sensitive to H-NSTEPEC antagonism than its chromosomal counterpart. H-NSR27 plays a relevant role by modulating R27 conjugation in response to temperature. Hence, we suggest that this modulator has evolved to avoid the deregulation of R27 conjugation by H-NSTEPEC-like proteins.

JTD Keywords: H-NS, Conjugation, R27, H-NS antagonism, H-NSTEPEC

Horizontal gene transfer (HGT), non-hereditary transfer of genetic material between organisms, accounts for a significant proportion of the genetic variability in bacteria. In Gram negative bacteria, the nucleoid-associated protein H-NS silences unwanted expression of recently acquired foreign DNA. This, in turn, facilitates integration of the incoming genes into the regulatory networks of the recipient cell. Bacteria belonging to the family Enterobacteriaceae express an additional protein, the Hha protein that, by binding to H-NS, potentiates silencing of HGT DNA. We provide here an overview of Hha-like proteins, including their structure and function, as well as their evolutionary relationship. We finally present available information suggesting that, by expressing Hha-like proteins, bacteria such as Escherichia coli facilitate HGT incorporation and hence, the impact of HGT in their genetic diversity.

JTD Keywords: Hha, H-NS, HGT DNA, Enterobacteria, Nucleoid-associated proteins, Enterica serovar typhimurium, Histone-like protein, h-ns, Escherichia-coli, Yersinia-enterocolitica, Salmonella-enterica

Proteins of the Hha/YmoA family co-regulate with H-NS the expression of virulence factors in Enterobacteriaceae. Vibrio cholerae lacks Hha-like proteins and its H-NS (vcH-NS) is unable to bind Hha, in spite of the conservation of a key residue for Hha binding by Escherichia coli H-NS (ecH-NS). Exchange of the residues in position 9 between vcH-NS and ecH-NS strongly reduces Hha binding by ecH-NS and introduces it in vcH- NS. These mutations strongly affect the repression of the hemolysin operon in E. coli and the electrophoretic mobility of complexes formed with a DNA fragment containing its regulatory region.

JTD Keywords: Nucleoid associated protein, H-NS, Hha, Transcription repression, NMR, Electrophoretic mobility shift assays

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

JTD Keywords: Nucleoid-associated protein, H-NS, Hha, Transcription repression

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.

JTD Keywords: H-NS, Dielectric behaviour, Hemolysin genes, Cells, Separation, Expression, Proteins, HHA, Electrorotation, Polarization