YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes. We also demonstrate that YdgT interacts both with H-NS and with StpA. Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell. The StpA protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.     

Functional replacement of the oligomerization domain of H-NS by the Hha protein of Escherichia coli

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of Escherichia coli H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the hns-induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.     

Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue.     

Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.     

The putative Orf4 of broad-host-range conjugative plasmid R446 could Be related to the H-NS family of bacterial nucleoid-associated proteins

The iml determinant of IncM plasmid R446 was described initially as a short fragment of DNA causing (when cloned in multicopy plasmids) insensitivity to pilus-dependent bacteriophage lysis in bacteria harboring coresident IncM plasmids. We have performed a computational analysis of the iml determinant of IncM plasmid R446 and found that a potential polypeptide (Orf4) shows similarity to the H-NS family of nucleoid-associated modulators of gene expression. The predicted protein has a molecular weight of 16.3 kDa and an isoelectric point of 5.50. Orf4 is adjacent and upstream of Orf5, a potential polypeptide related to the Hha/YmoA/RmoA family of proteins which modulate bacterial virulence expression (Hha and YmoA) and R100 transfer (RmoA) in response to environmental conditions.     

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli. Received. 18 October 1999 / Accepted: 21 December 1999     

A cyanobacterial gene family coding for single-helix proteins resembling part of the light-harvesting proteins from higher plants.

In the cyanobacterium Synechocystis sp. PCC 6803 five genes were identified with significant sequence similarity to regions of members of the eukaryotic chlorophyll a/b binding gene family (Cab family) and to hliA, a gene coding for a small high-light-induced protein in Synechococcus sp. PCC 7942. Four of these five genes are 174-213 bp in length and code for small proteins predicted to have a single transmembrane helix. The fifth Cab-like gene in Synechocystis sp. PCC 6803 is much longer and codes for a protein of which the N-terminal 80% resemble ferrochelatase but the C-terminal domain has similarity to Cab regions. The small genes were expressed preferentially in the absence of photosystem I, but gene expression was not significantly enhanced at moderately high light intensity. Therefore they were not designated as hli (high-light-induced) as was done for the Synechococcus sp. PCC 7942 homolog. Instead, the genes have been named scp, as the corresponding polypeptides of Synechocystis sp. PCC 6803 are small Cab-like proteins (SCP). The scpA gene, which codes for ferrochelatase with a C-terminal Cab-like extension, was interrupted by the insertion of a kanamycin-resistance cassette between the ferrochelatase and Cab-like gene domains. In the PS I-less background, interruption of scpA was found to lead to increased tolerance to high light intensity and to the requirement of a slightly higher light intensity to drive photosystem II electron transfer, suggestive of decreased light-harvesting efficiency in the absence of the C-terminal extension of ScpA. Immunodetection of ScpC and ScpD indicated that either or both accumulated in PS I-less strains. These proteins were also detected in bands of more than 45 kDa on denaturing gels, raising the possibility that they may occur as stable oligomers. The SCPs represent a new group of cyanobacterial proteins that, in view of their primary structure and response to deletion of photosystem I, are likely to be involved in transient pigment binding.     

Role of proteins of the macroglobulin family in the mechanisms of infection

Information on the properties of proteins of the macroglobulin family taking part in the host protection from viral, fungal and bacterial pathogens is reviewed. High plasticity and polyfunctional character of these proteins makes it possible to realize different protective functions. They inhibit the lysis of the cell wall by binding the hydrolases of the pathogen thus blocking its penetration into the cell, directly participate in the presentation of antigens to immunocompetent cells, transport antibacterial substances (interferons, lysozyme) to the zone of infection. In addition, macroglobulins take part in the apoptosis regulation in infected cells, utilization of the lysosomal enzymes of annihilated pathogens. The complexes of macroglobulins with some proteins are powerful inductors of antibody production. Further studies of the properties of these proteins will result in a better understanding of the nature of infectious process. The possibility of artificial formation of macroglobulin complexes with pathogen components or with substances possessing protective or anti-inflammatory properties opens prospects for using these proteins in the fields of vaccinology, gene therapy and molecular biology.     

Cnu, a novel oriC-binding protein of Escherichia coli

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.     

Plant cells contain a novel member of the retinoblastoma family of growth regulatory proteins.

The product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. Animal virus oncoproteins interact with the Rb protein via an LXCXE motif and disrupt Rb-E2F complexes, driving cells into S-phase. Recently, we found that the RepA protein of a plant geminivirus contains an LXCXE motif that is essential for its function, a finding that predicts the existence of Rb-related proteins in plant cells. Here we report the isolation of a maize cDNA clone encoding a protein (ZmRb1) which, based on structural and functional studies, is closely related to the mammalian Rb family of growth regulatory proteins. ZmRb1 shows a high degree of amino acid conservation when compared with animal Rb members, particularly in the A/B 'pocket' domain, but ZmRb1 has a shorter N-terminal domain. ZmRb1 forms stable complexes with plant LXCXE-containing proteins, e.g. geminivirus RepA protein. Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the Rb family. This suggests that ZmRb1 controls the G1/S transit in plant cells and is consistent with the fact that geminiviruses need an S-phase environment for DNA replication, as animal DNA tumor viruses do. Our results allow the extension of the Rb family of tumor suppressor proteins to plants and have implications on animal and plant strategies for cell growth control.     

A new class of proteins regulating gene expression in enterobacteria

YmoA and Hha are highly similar bacterial proteins downregulating gene expression In Yersinia entero-colitlca and Escherichia coli. respectively. The pheno-type of ymoA mutants evokes that of mutants affected in some histone-like proteins. This paper describes complementation of a ymoA mutation in Y. enterocolitica by the hha gene from E. coli. We show that YmoA and Hha are not only very similar proteins but that they are functionally Interchangeable. Genetic experiments indicate that Hha can also stimulate transposition events in vivo. By Southern Biol analysis we detected hha-homologous genes at least in Citrobacter diversus, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Salmonella typhimurium. We suggest that both YmoA and Hha belong to a new family of proteins down regulating gene expression in different enterobacteria.     

Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.