SummaryBacterial cytokinesis requires the divisome, a complex of proteins that co-ordinates the invagination of the cytoplasmic membrane, inward growth of the peptidoglycan layer and the outer membrane. Assembly of the cell division proteins is tightly regulated and the order of appearance at the future division site is well organized. FtsQ is a highly conserved component of the divisome among bacteria that have a cell wall, where it plays a central role in the assembly of early and late cell division proteins. Here, we describe the crystal structure of the major, periplasmic domain of FtsQ from Escherichia coli and Yersinia enterocolitica. The crystal structure reveals two domains; the a-domain has a striking similarity to polypeptide transport-associated (POTRA) domains and the C-terminal b-domain forms an extended b-sheet overlaid by two, slightly curved a-helices. Mutagenesis experiments demonstrate that two functions of FtsQ, localization and recruitment, occur in two separate domains. Proteins that localize FtsQ need the second b-strand of the POTRA domain and those that are recruited by FtsQ, like FtsL/FtsB, require the surface formed by the tip of the last a-helix and the two C-terminal b-strands. Both domains act together to accomplish the role of FtsQ in linking upstream and downstream cell division proteins within the divisome.

We have developed a stable isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI q gene from pYanni3 and the lac promoter, P A1/04/03 , from pJBA24.Rhodobacter sphaeroides and Paracoccus denitrificans are often used for the study of bacterial metabolism, bioenergetics (8), and signal transduction (11). Although inducible-expression plasmids are available for these organisms, e.g., pRKSK1 (5) and pRECTX (9), these plasmids suffer from one or more of the following problems. First, continuous antibiotic selection is essential for maintaining the plasmid in the population, e.g., plasmid pRK415 (7), which is the vector backbone for most of the available expression vectors for these species, is retained by only approximately 10% of the population after 40 generations without antibiotic selection (6) (segregational instability). Second, the inducer affects the expression of many endogenous genes; for example, several R. sphaeroides vectors use either light-or oxygeninducible promoters to deliver high levels of protein expression (5). However, light and oxygen affect the expression of over 35% of the endogenous genes in this organism (2), which limifts the use of these vectors in functional studies.The plasmid developed in this study, pIND4 ( Fig. 1), uses the pMG170 vector backbone (6), the lacI q gene from pYanni3 (4), and the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible-expression cassette from pJBA24, which includes the P A1/04/03 promoter, a ribosome binding site, a polylinker, and two transcriptional terminators (1). pMG170 confers kanamycin resistance and is an Escherichia coli shuttle cloning vector derived from naturally occurring plasmid pMG160 of Rhodobacter blasticus, which replicates and is segregationally stable in several other members of the Rhodobacteraceae (6). In E. coli, pMG170 has a high copy number, replicating using the ColE1 origin, while in R. sphaeroides, the pMG160 origin delivers a copy number of 18 to 23 (6).Vector construction. The construction of pIND4 is described in the supplemental material.Testing of the expression plasmid. The coding sequence for the R. sphaeroides cheY6 gene was cloned into pIND4, generating pIND4-Y6. The plasmid was introduced into R. sphaeroides strain JPA1336 (⌬cheY6 derivative of WS8N) and P. denitrificans strain PD1222 (wild type) via conjugation with the E. coli donor strain S17-1 pir (10). Cells containing the plasmid were grown from single colonies under aerobic conditions with shaking (225 rpm) in succinate medium containing 25 g/ml kanamycin. The effects of different concentrations of IPTG (Fig. 2) and different induction times (Fig. 3) on CheY6 protein accumulation were investigated.At IPTG concentrations of less than 1 M, no expression of CheY6 was detectable in R. sphaeroides (the minimum detection l...

Aims:  To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. Methods and Results:  Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence‐associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O‐antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS‐dependent phages. Conclusions:  Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. Significance and Impact of the Study:  This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.

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