Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens

Abstract

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding proteindependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacter jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells. Infections of wound sites in dicotyledonous plants byAgrobacterium tumefaciens result in the formation of crown gall tumors. The mechanism of pathogenesis involves the transfer of a DNA fragment (T-DNA) from the bacterial Ti plasmid to the plant host cell (4). An early step in tumor formation is attachment of bacteria to the plant cell surface (15). This attachment is required for pathogenesis since all known nonattaching mutants are avirulent (3,5,16,25). We have previously isolated three Tn5 nonattaching mutants (16). These mutants all mapped to the same region of the bacterial chromosome (close to met6) and contained Tn5 insertions in a large EcoRI fragment (22). In order to characterize the process of bacterial attachment and to identify the genes involved, we examined this region of chromosomal DNA in greater detail. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study were as previously described (14,16,19). Bacteria were grown in Luria broth, nutrient broth, or minimal medium, and viable cell counts were determined as previously described (14). A. tumefaciens was grown at 25ЊC. Escherichia coli was grown at 37ЊC. Antibiotics were used at the following concentrations: carbenicillin, 50 mg/liter; tetracycline, 10 mg/liter; chloramphenicol, 100 mg/liter; neomycin, 20 mg/liter for liquid media and 60 mg/liter for agar media; and gentamycin, 50 mg/liter. A library of A. tumefaciens NT1 DNA cloned as Sau3a partial digestion products into the BamHI site of pCP13 was obtained from S. Farrand. A library of A. tumef...

Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms

“…This defect can be rescued in a dosedependent manner by supplementation of the growth medium with putrescine, suggesting that both exogenous and endogenous putrescine can activate biofilm formation (249). Furthermore, spermidine and putrescine transporters have been implicated in surface-associated growth of Agrobacterium tumefaciens and Pseudomonas putida (216,280). Taken together, these findings suggest that polyamines may be regulators of surface-associated growth and biofilm formation in diverse bacteria.…”

“…Another exopolysaccharide that is commonly found in biofilm matrices is cellulose, a linear polymer of (1-4)-␤-linked glucose. Cellulose is a major component of the biofilm matrices of some E. coli strains and of some species of Salmonella, Citrobacter, Enterobacter, and Pseudomonas as well as Agrobacterium tumefaciens (66,216,297,299,327,362,363). In E. coli and S. Typhimurium, the synthesis of cellulose is carried out by the proteins encoded by the bacterial cellulose synthesis operons, bcsABZC-bcsEFG (297,363).…”

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

“…In Agrobacterium tumefasciens and Vibrio cholerae, homologs of polyamine transport (Pot) membrane proteins are important for surface attachment (Karatan et al 2005;Matthysse et al 1996). Although the Y. pestis polyamine transport genes are highly induced in the flea, mutational loss of this system did not affect biofilm formation in the flea, ruling out an essential role for these proteins in adherence, but the polyamine biosynthesis genes of this pot mutant were intact (Vadyvaloo et al 2007).…”

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

“…We found a cluster of genes (529,782-537,888) whose products are 58-70% identical to A. tumefaciens proteins required for attachment to plant cells (60). attABC encode a putative ABC transporter, and atrABC encode proteins similar to a transcriptional regulator, glutamate-1-semialdehyde 2,1-aminomutase, and acetolactate synthase, respectively (55).…”