The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, ot and 0, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a pertubation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition.The Escherichia coli HU protein is the most abundant DNA-binding protein present in the bacterial cell (38). This small, basic, dimeric protein interacts with double-stranded DNA, single-stranded DNA, and RNA. As with mixtures of the four core eucaryotic histones, HU can introduce negative supercoils into a relaxed closed circular DNA in vitro if topoisomerase I is present, and the resulting condensed structures resemble nucleosomes (5, 40). In addition, just as the histones in eucaryotes are conserved, the HU protein is highly conserved among procaryotic organisms and also in mitochondria and chloroplasts (10). It was shown earlier that HU is associated with the E. coli nucleoid (37, 47) (although some cytological observations were interpreted as locating HU on the edge rather than inside of the nucleoid [11]), but whether it has a role in maintaining the structure of the chromosome as a whole is not known.Biochemical studies have suggested a role for HU in the site-specific recognition of nucleotide sequences by other proteins (for a review, see reference 10). Among these interactions are the formation of protein-DNA complexes involved in the initiation of replication of the bacterial chromosome at oriC, in the hin-mediated gene inversion, and in the transposition of transposon TnJO and bacteriophage Mu. More precisely, replication from oriC (9, 13) and the transposition of TnJO (29,34) are moderately stimulated by HU protein, whereas the flagellar phase variation in Salmonella typhimurium (21) MATERIALS AND METHODSStrains and bacteria. The bacterial strains, plasmids, and phages used in this study are listed in Table 1. Bacteria were grown in LB (10 g of Bactotryptone [Difco Laboratories], 5 g of yeast extract, and 5 g of NaCl per liter) and counted on L agar (LA) (28). Minimal medium was M9 supplemented with thiamine (10 ,ug/ml), amino acids (100 pg/ml) or casamino acids (0.4%, wt/vol), and 0.4% (wt/vol) sugar (glycerol, maltose, lactose, or glucose). Kanamycin (25 p.g/ml), chloramphenicol (12.5 pLg/ml), streptomycin (200 pig/ml), and spectinomycin (100 ,ug/ml) were included when appropriate.Methods for Mu assays. The general methods used to manipulate the phage have been described previously (6,12 (ii) Mu transposition assay. The frequency of transposition onto the pUZ8 plasmid was measured by a mating-out assay. The Mu cts62pApl lysogens (or the bacteria infected with the bacteriophage X mini-Mu Apr) carrying pUZ8 were mated with the MFG625B recipient on a filter. After incubation for 2 h at 42...

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SummaryIt is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.

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The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.

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