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RsmC and FlhDC are global regulators controlling extracellular proteins/enzymes, rsmB RNA, motility, and virulence of Erwinia carotovora subsp. carotovora . FlhDC, the master regulator of flagellar genes, controls these traits by positively regulating gacA , fliA , and rsmC and negatively regulating hexA . RsmC, on the other hand, is a negative regulator of extracellular proteins/enzymes, motility, and virulence since the deficiency of RsmC in FlhDC + strain results in overproduction of extracellular proteins/enzymes, hypermotility, and hypervirulence. These phenotypes are abolished in an RsmC − FlhDC − double mutant. We show that RsmC interferes with FlhDC action. Indeed, the expression of all three targets (i.e., gacA , rsmC , and fliA ) positively regulated in E . carotovora subsp. carotovora by FlhDC is inhibited by RsmC. RsmC also partly relieves the inhibition of hexA expression by FlhDC. The results of yeast two-hybrid analysis revealed that RsmC binds FlhD and FlhDC, but not FlhC. We propose that binding of RsmC with FlhD/FlhDC interferes with its regulatory functions and that RsmC acts as an anti-FlhD 4 FlhC 2 factor. We document here for the first time that RsmC interferes with activation of fliA and motility in several members of the Enterobacteriaceae family. The extent of E . carotovora subsp. carotovora RsmC-mediated inhibition of FlhDC-dependent expression of fliA and motility varies depending upon enterobacterial species. The data presented here support the idea that differences in structural features in enterobacterial FlhD are responsible for differential susceptibility to E . carotovora subsp. carotovora RsmC action.

In this study, utilizing a Corynebacterium glutamicum Δ pimB ′ Δ mgtA double deletion mutant, we unequivocally assign the in vivo functions of Rv2188c as an Ac 1 PIM 1 :mannosyltransferase (originally termed PimB′ Mt [ Mycobacterium tuberculosis PimB′]) and Rv0557 as a GlcAGroAc 2 :mannosyltransferase (originally termed PimB Mt ), which we have reassigned as PimB Mt and MgtA Mt , respectively, in Mycobacterium tuberculosis .

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N -acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi ; LrhA [LysR homolog A] in Escherichia coli ) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC − mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin Ecc ) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC + plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora , like those of other enterobacteria, consists of flhD and flhC . Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA , rsmC , and fliA and negatively regulates hexA ( lrhA ). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA . Reduced levels of GacA and elevated levels of HexA in the FlhDC − mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA − FlhDC − double mutant and multiple copies of rsmB + DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora . Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora .

N -acyl homoserine lactone (AHL) is required by Erwinia carotovora subspecies for the expression of various traits, including extracellular enzyme and protein production and pathogenicity. Previous studies with E. carotovora subsp. carotovora have shown that AHL deficiency causes the production of high levels of RsmA, an RNA binding protein that functions as a global negative regulator of extracellular enzymes and proteins and secondary metabolites (Rsm, regulator of secondary metabolites). We document here that ExpR, a putative AHL receptor belonging to the LuxR family of regulators, activates RsmA production. In the absence of AHL, an ExpR + E. carotovora subsp. carotovora strain compared to its ExpR − mutant, produces higher levels of rsmA RNA and better expresses an rsmA-lacZ transcriptional fusion. Moreover, the expression of the rsmA-lacZ fusion in Escherichia coli is much higher in the presence of expR 71 (the expR gene of E. carotovora subsp. carotovora strain Ecc71) than in its absence. We also show that purified preparation of MBP-ExpR 71 binds (MBP, maltose binding protein) rsmA DNA. By contrast, MBP-ExpR 71 does not bind ahlI (gene for AHL synthase), pel-1 (gene for pectate lyase), or rsmB (gene for regulatory RNA that binds RsmA), nor does ExpR 71 activate expression of these genes. These observations strongly suggest transcriptional activation of rsmA resulting from a direct and specific interaction between ExpR 71 and the rsmA promoter. Several lines of evidence establish that N -3-oxohexanoyl- l -homoserine lactone (3-oxo-C6-HL), the major AHL analog produced by E. carotovora subsp. carotovora strain Ecc71, inhibits ExpR 71 -mediated activation of rsmA expression. These findings for the first time establish that the expR effect in E. carotovora subsp. carotovora is channeled via RsmA, a posttranscriptional regulator of E. carotovora subspecies, and AHL neutralizes this ExpR effect.

RsmA (for regulator of secondary metabolism), RsmC, and rsmB RNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity in Erwinia carotovora subsp . carotovora. RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay. rsmB RNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA and rsmB RNA by positively regulating rsmA and negatively controlling rsmB. The level of rsmB RNA is substantially higher in RsmA + bacteria than in RsmA − mutants. We show that rsmB RNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of an rsmB-lacZ transcriptional fusion; in fact, the β-galactosidase level is somewhat higher in RsmA − bacteria than in RsmA + bacteria. We also investigated the basis for increased levels of rsmA and rsmB RNAs in the absence of the quorum-sensing signal, N -[3-oxohexanoyl]- l -homoserine lactone (OHL). The absence of OHL activates transcription of rsmA but not of rsmB. Instead, increased stability of rsmB RNA in the presence of RsmA accounts for the elevated levels of the rsmB RNA in OHL − bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels of rsmB RNA, high levels of rsmB RNA occur in the absence of RsmC or OHL only in RsmA + bacteria, indicating a critical role for RsmA in modulating the levels of rsmB RNA. The findings reported here firmly establish that the quorum-sensing signal is channeled in E. carotovora subsp. carotovora via the rsmA-rsmB posttranscriptional regulatory system.

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin Ecc (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB , a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219–234, 1998). We have cloned and characterized a novel regulatory gene, rsmC , that activates RsmA production and represses extracellular enzyme and harpin Ecc production, rsmB transcription, and virulence in E. carotovora subsp. carotovora . In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA + allele, the basal levels of Pel, Peh, Cel, Prt, and harpin Ecc as well as the amounts of rsmB , pel-1 , peh-1 , celV , and hrpN Ecc transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC − mutant than in the RsmC + parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC − mutant than in the RsmC + parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC − mutant, an RsmA − mutant, and an RsmA − RsmC − double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB . Exoprotein production by the RsmC − mutant is partially dependent on the quorum sensing signal, N -(3-oxohexanoyl)- l -homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica , E. carotovora subsp. betavasculorum , and E. carotovora subsp. carotovora . These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces Harpin Ecc , the elicitor of the hypersensitive reaction. We document here that KdgR Ecc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgR Ech and the Escherichia coli repressor, KdgR Eco , negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and Harpin Ecc production in E. carotovora subsp. carotovora . The levels of pel-1 , peh-1 , celV , and hrpN Ecc transcripts are markedly affected by KdgR Ecc . The KdgR Ecc − mutant is more virulent than the KdgR Ecc + parent. Thus, our data for the first time establish a global regulatory role for KdgR Ecc in E. carotovora subsp. carotovora . Another novel observation is the negative effect of KdgR Ecc on the transcription of rsmB (previously aepH ), which specifies an RNA regulator controlling exoenzyme and Harpin Ecc production. The levels of rsmB RNA are higher in the KdgR Ecc − mutant than in the KdgR Ecc + parent. Moreover, by DNase I protection assays we determined that purified KdgR Ecc protected three 25-bp regions within the transcriptional unit of rsmB . Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgR Ecc -binding site as 5′-G/AA/TA/TG AAA [N 6 ] TTT CAG/TG/TA-3′. Two such KdgR Ecc -binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgR Ecc -binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB . KdgR Ecc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgR Ecc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgR Ecc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.

RpoS (sigma-S or sigma-38) controls a large array of genes that are expressed during stationary phase and under various stress conditions in Escherichia coli and other bacteria. We document here that plant pathogenic and epiphytic Erwinia species, such as E. amylovora ; E. carotovora subsp. atroseptica , betavasculorum , and carotovora ; E. chrysanthemi ; E. herbicola ; E. rhapontici ; and E. stewartii , possess rpoS genes and produce the alternate sigma factor. We show that rpoS transcription in E. carotovora subsp. carotovora is driven from a major promoter which resides within the nlpD gene located upstream of rpoS as in E. coli . RpoS − E. carotovora subsp. carotovoa strain AC5061, constructed by marker exchange, is more sensitive to hydrogen peroxide, carbon starvation, and acidic pH than its RpoS + parent strain, AC5006. The basal levels of extracellular pectate lyase, polygalacturonase, and cellulase as well as those of transcripts of E. carotovora subsp. carotovora hrpN ( hrpN Ecc ), the gene for the elicitor of the hypersensitive reaction, are higher in the RpoS − strain than in the RpoS + parent. Likewise, compared to AC5006, AC5061 causes more extensive maceration of celery petioles. Our findings with the RpoS − mutant and strains carrying multiple copies rpoS + DNA reveal that rpoS positively controls rsmA expression. We also present evidence that supports the hypothesis that the RpoS effect on extracellular enzyme levels, hrpN Ecc expression, and virulence manifests itself by the modulation of rsmA expression.

The production of extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) is activated by the cell density (quorum)-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (HSL); plant signals; and aep genes during postexponential growth of Erwinia carotovora subsp. carotovora 71. Studies with mutants of E. carotovora subsp. carotovora 71 derepressed in exoenzyme production led to the identification of a negative regulator gene, rsmA (rsm, repressor of secondary metabolites). Nucleotide sequencing, transcript assays, and protein analysis established that a 183-bp open reading frame encodes the 6.8-kDa RsmA. rsmA has extensive homology with the csrA gene of Escherichia coli, which specifies a negative regulator of carbon storage. Moreover, the suppression of glycogen synthesis in E. coli by rsmA indicates that the Erwinia gene is functionally similar to csrA. Southern hybridizations revealed the presence of rsmA homologs in soft-rotting and non-soft-rotting Erwinia spp. and in other enterobacteria such as Enterobacter aerogenes, E. coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, and Yersinia pseudotuberculosis. rsmA suppresses production of Pel, Peh, Cel, and Prt, plant pathogenicity, and synthesis of HSL in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, and E. chrysanthemi. In the E. carotovora subsp. carotovora 71, rsmA reduces the levels of transcripts of hslI, a luxI homolog required for HSL biosynthesis. This specific effect and the previous finding that HSL is required for extracellular enzyme production and pathogenicity in soft-rotting Erwinia spp. support the hypothesis that rsmA controls these traits by modulating the levels of the cell density (quorum)-sensing signal.

In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.

The N -acylhomoserine lactone (AHL) signaling system comprises a producing system that includes acylhomoserine synthase (AhlI, a LuxI homolog) and a receptor, generally a LuxR homolog. AHL controls exoprotein production in Erwinia carotovora and consequently the virulence for plants. In previous studies we showed that ExpR, a LuxR homolog, is an AHL receptor and that it activates transcription of rsmA , the gene encoding an RNA binding protein which is a global negative regulator of exoproteins and secondary metabolites. An unusual finding was that the transcriptional activity of ExpR was neutralized by AHL. We subsequently determined that the genomes of most strains of E. carotovora subspecies tested possess two copies of the expR gene: expR1 , which was previously studied, and expR2 , which was the focus of this study. Comparative analysis of the two ExpR variants of E. carotovora subsp. carotovora showed that while both variants activated rsmA transcription, there were significant differences in the patterns of their AHL interactions, the rsmA sequences to which they bound, and their relative efficiencies of activation of rsmA transcription. An ExpR2 − mutant produced high levels of exoproteins and reduced levels of RsmA in the absence of AHL. This contrasts with the almost complete inhibition of exoprotein production and the high levels of RsmA production in an AhlI − mutant that was ExpR1 − . Our results suggest that ExpR2 activity is responsible for regulating exoprotein production primarily by modulating the levels of an RNA binding protein.

RsmA (for regulator of secondary metabolism), RsmC, and rsmB RNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity in Erwinia carotovora subsp. carotovora. RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay. rsmB RNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA and rsmB RNA by positively regulating rsmA and negatively controlling rsmB. The level of rsmB RNA is substantially higher in RsmA(+) bacteria than in RsmA(-) mutants. We show that rsmB RNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of an rsmB-lacZ transcriptional fusion; in fact, the beta-galactosidase level is somewhat higher in RsmA(-) bacteria than in RsmA(+) bacteria. We also investigated the basis for increased levels of rsmA and rsmB RNAs in the absence of the quorum-sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone (OHL). The absence of OHL activates transcription of rsmA but not of rsmB. Instead, increased stability of rsmB RNA in the presence of RsmA accounts for the elevated levels of the rsmB RNA in OHL(-) bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels of rsmB RNA, high levels of rsmB RNA occur in the absence of RsmC or OHL only in RsmA(+) bacteria, indicating a critical role for RsmA in modulating the levels of rsmB RNA. The findings reported here firmly establish that the quorum-sensing signal is channeled in E. carotovora subsp. carotovora via the rsmA-rsmB posttranscriptional regulatory system.

Cells of Escherichia coli take up vitamin B 12 (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella . The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His 6 tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF , but not in the flanking genes pfs and yadS , strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC , btuD , or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.

R plasmid R68.45 was transferred in broth matings from Escherichia coli to strains of Erwinia amylovora, E. carotovora subsp. atroseptica, E. chrysanthemi , and E. herbicola ( Enterobacter agglomerans ); the frequency of transfer ranged from 2 × 10 −8 to 5 × 10 −4 per input donor cell depending on the bacterial species. The drug resistance markers tet + , amp + , and kan + were stable in these Erwinia species. Transconjugants of Erwinia spp., but not of the wild-type parent Erwinia strains, acquired levels of antibiotic resistance (tetracycline, 50 μg/ml; ampicillin, 200 μg/ml; kanamycin 200 μg/ml) similar to those of the donor R68.45-bearing strain of Escherichia coli. Erwinia transconjugants (with one exception of E. carotovora subsp. atroseptica ) were donors of the antibiotic resistance markers; the frequency of transfer was consistently higher with an E. coli strain than with Erwinia spp. as recipients, and when matings were done on a solid surface (membranes) rather than in liquid. Transfer of chromosomal markers ade + , gal + , gtu + (utilization of galacturonate), his + , leu + , lys + , thr + , and trp + occurred in crosses between E. chrysanthemi strains harboring R68.45 and appropriate recipient strains; the frequency of transfer ranged from 9.0 × 10 −8 to 2.0 × 10 −6 depending on the selective marker. Analysis of the coinheritance of unselected markers among various classes of recombinants revealed linkage between thr-leu-lys-ade and between trp and his , thus confirming earlier findings with the Hfr-type donor cells. Since R68.45 mobilized an array of chromosomal markers in the wild-type as well as genetically marked strains of E. chrysanthemi , the system, used in conjunction with the existing Hfr strains, should provide a useful tool to study the genetics of plant pathogenicity of this bacterial species. In contrast to E. chrysanthemi , R68.45 did not mobilize chromosomal markers ilv + , his + , rbs + , ser + , and thr + in E. amylovora EA178.

Stable donor strains of Erwinia amylovora were obtained from strain EA178R 1 (harboring an Escherichia coli F′ lac ) by selection for clones resistant to curing by acridine orange. These donor strains (EA178R 1 -99 and EA178R 1 -111) transfer chromosomal markers ( arg, cys, gua, ilv, met, pro, ser, trp ); the frequency of the appearance of recombinants prototrophic for Cys, Gua, Met, Ser, and Trp is highest (> 10 −5 ), followed by recombinants prototrophic for Arg, Ilv, and Pro (10 −7 to 10 −5 ). The results of interrupted matings, as well as the frequency of transmission of various markers, suggest that cys is transferred as an early marker by both donor strains. The Hfr state of these donor strains is rather likely on the basis of the following observations. The donor strains exhibit a relatively efficient and possibly oriented chromosome transfer; the Lac + character is not cured by acridine orange in these donor strains; and these donor strains do not transfer F.

Antibiotic resistance carried on R factors was transferred by conjugation from Escherichia coli B/r and Shigella flexneri 1a to Erwinia spp. Tetracycline resistance (TetR) carried on R factor R100 drd-56 was transferred from E. coli B/r to strains of Erwinia amylovora, E. aroideae, E. atroseptica, E. chrysanthemi, E. cytolytica, E. dissolvens, E. herbicola, E. nigrifluens , and E. nimipressuralis , but not to strains of Erwinia carotovora, E. carnegieana, E. dieffenbachiae, E. oleraceae , and E. quercina . Multiple antibiotic resistance (chloramphenicol, streptomycin, tetracycline; ChlR-StrR-TetR) carried on R factor SR1 was transferred from a clinical isolate of S. flexneri 1a to strains of E. aroideae, E. chrysanthemi, E. herbicola , and E. nigrifluens , but not to strains of other Erwinia spp. The frequency of this transfer was low with receptive cultures of Erwinia spp. and E. coli (F − strain). Antibiotic resistance in the exconjugants showed varying degrees of stability in the presence or absence of acridine orange, depending on the strain tested. The frequencies of segregation to drug susceptibility in the presence of acridine orange, though low, suggest that the elements exist as plasmids in the majority of the Erwinia exconjugants. Multiple antibiotic resistance (ChlR-StrR-TetR) was found to segregate into various resistance classes (ChlR-StrR, StrR-TetR, TetR, StrR, and none) in these exconjugants. The exconjugants of E. amylovora, E. herbicola , and E. nigrifluens , to which R100 drd-56 was transferred from E. coli B/r, were sensitive to the male (F)-specific phage M13. There was a positive correlation between the susceptibility of exconjugants to the F-specific phage M13 and their ability to transfer R100 drd-56 to the recipient cultures of Escherichia coli, Erwinia herbicola, Salmonella typhimurium , and Shigella dysenteriae . Exceptions were, however, noted with Erwinia dissolvens and E. nimipressuralis exconjugants harboring R100 drd-56 ; these exconjugants, although not susceptible to M13, transferred R100 drd-56 to the recipient cultures. The frequency of transfer of R100 drd-56 and the levels of resistance to tetracycline in Erwinia exconjugants were found to differ markedly depending upon the strain employed. Transfer of multiple antibiotic resistance (ChlR-StrR-TetR) from Erwinia exconjugants was not obtained in preliminary trials with an E. coli F − strain as the recipient culture.