Phage Receptor Sites Research Articles

Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Adsorption of temperate phages ofLactobacillus delbrueckiistrains and phage resistance linked to their cell diversity

The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage-resistant derivatives with interesting technological properties. The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca2+ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage-resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage-resistant derivatives of their host strains were obtained.

A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.

Diversity of phage types among archived cultures of the Demerec collection of Salmonella enterica serovar Typhimurium strains.

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.

Bacteriophage lambda receptor site on the Escherichia coli K-12 LamB protein.

We have analyzed eight new phage-resistant missense mutations in lamB. These mutations identify five new amino acid residues essential for phage lambda adsorption. Two mutations at positions 245 and 382 affect residues which were previously identified, but lead to different amino acid changes. Three mutations at residues 163, 164, and 250 enlarge and confirm previously proposed phage receptor sites. Two different mutations at residue 259 and one at 18 alter residues previously suggested as facing the periplasmic face. The mutation at residue 18 implicates for the first time the amino-terminal region of the LamB protein in phage adsorption. The results are discussed in terms of the topology of the LamB protein.

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease.

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.

Further sequence analysis of the phage lambda receptor site: Possible implications for the organization of the LamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.

Apparent bacteriophage-binding region of an Escherichia coli K-12 outer membrane protein.

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli. It serves as the receptor for several T-even-like phages and is required for the action of certain colicins and for the stabilization of mating aggregates in conjugation. We have isolated two mutant alleles of the cloned ompA gene which produce a protein that no longer functions as a phage receptor. Bacteria possessing the mutant proteins were unable to bind the phages, either reversibly or irreversibly. However, both proteins still functioned in conjugation, and one of them conferred colicin L sensitivity. DNA sequence analysis showed that the phage-resistant, colicin-sensitive phenotype exhibited by one mutant was due to the amino acid substitution Gly leads to Arg at position 70. The second mutant, which contained a tandem duplication, encodes a larger product with 8 additional amino acid residues, 7 of which are a repeat of the sequence between residues 57 and 63. In contrast to the wild-type OmpA protein, this derivative was partially digested by pronase when intact cells were treated with the enzyme. The protease removed 64 NH2-terminal residues, thereby indicating that this part of the protein is exposed to the outside. It is argued that the phage receptor site is most likely situated around residues 60 to 70 of the OmpA protein and that the alterations characterized have directly affected this site.

Use of Lectins to Characterize the Receptor Sites for Bacteriophage PL-1 of Lactobacillus casei

SUMMARY: The polysaccharide (PS) located outside the peptidoglycan layer in Lactobacillus casei ATCC 27092 was found to inhibit the adsorption of PL-1 phage to cell wall preparations without inactivating free phage. Electron microscopic examination of adsorption mixtures showed that the phage were adsorbed to fragments of PS material in a tail-first orientation. Phage did not adsorb to isolated peptidoglycan. The PS was composed of l-rhamnose, d-glucose, d-glucosamine and d-galactosamine, with the hexosamines possibly in N-acetylated form. Prior treatment of L. casei with Streptomyces haemagglutinin, an anti-human blood group B agglutinin that binds specifically to l-rhamnose, resulted in a concentration-dependent inhibition of phage adsorption. Phage adsorption was inhibited partially by lectins specific for d-glucose and N-acetyl-d-glucosamine, but not by lectins specific for N-acetyl-d-glucosamine or N-acetyl-d-galactosamine. The inhibition of phage adsorption occurred immediately upon the addition of the effective lectins, and was reversed by the addition of the respective lectin inhibitors, α-phenyl galactoside or α-methyl glucoside. The results indicate that l-rhamnosyl residues are the main determinants of the PL-1 phage receptor sites, while d-glucosyl residues may be involved more indirectly.

Characterization of bacteriophage D6

Bacteriophage D6, described by Mise and Suzuki [ J. Virol. 6, 253–255 (1970)], is a temperate coliphage capable of generalized transduction. D6 resembles phage P1 morphologically and is slightly sensitive to anti-P1 serum. We have found that like P1, the D6 prophage is a plasmid, approximately 90 kb in size. The D6 prophage is compatible with both P1 and the closely related heteroimmune prophage P7, both of which are in incompatibility group Y. D6 DNA is restricted by the P1 and P7 prophages, but in the absence of restriction, D6 is able to grow on P1 and P7 lysogens. Neither P1 nor P7 is able to grow on a D6 lysogen. This is not due to DNA restriction or to the elimination of phage receptor sites on the host's surface. Therefore a nonreciprocal immunity relationship appears to exist between P1–P7 and D6. Mutants of P1 and P7 which are not subject to repression by the P1–P7 c 1 + gene product are able to grow on a D6 lysogen, implying that D6 has a repressor activity like that of P1 c 1. There are two regions of strong hybridization between P1 and D6 DNA; one includes the invertible C loop of Pl. Attempts to demonstrate complementation or recombination between P1 or P7 and D6 were not successful, even for markers in the regions of homology.

Caulobacter crescentus pili: structure and stage-specific expression.

Pili are functionally expressed during the predivisional and swarmer stages of the Caulobacter crescentus differentiation cycle. They appear on the developing swarmer pole and at the same cellular location as flagella and the phiCbK receptor sites. Pili disappear when the swarmer cell differentiates into a stalked cell; this occurs with the loss of flagella and the disappearance of phage receptor sites. C. crescentus CB13B1a pili have been purified and characterized. Monomeric pilin is a protein with an apparent molecular weight of 8,500 that stains weakly with periodic acid-Schiff reagent. The amino acid composition of purified pilin reveals very low quantities of basic amino acids and a complete absence of methionine. Pilin is synthesized throughout the C. crescentus differentiation cycle. Neither free pili nor pilin monomers are detectable in the growth media, suggesting that loss of piliation in the swarmer- to stalked-cell transition occurs via pilus retraction.

Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B.

Coliphage T4D was strongly adsorbed to intact lipopolysaccharides and alkaline and lipase-treated lipopolysaccharides from cells of Escherichia coli B, but was not so adsorbed to heat-treated cells. In contrast, coliphage T2h was not adsorbed to lipopolysaccharides and the heat-treated cells. Acid hydrolysate of lipopolysaccharides strongly inhibited the adsorption of phage T4D to acetone and ether-treated cells. The adsorption of phage T4D to the acetone and ether-treated cells was markedly inhibited by authentic D-glucosamine, N-acetyl-D-glucosamine, alpha-methyl-N-acetyl-D-glucosaminide, alpha-methyl-D-glucoside, and D-maltose. Authentic D-glucose and E,L-2,6-diaminopimelic acid also showed similar activity. These compounds did not affect the adsorption of phage T2h to the acetone- and ether-treated cells. Concanavalin A and wheat-germ agglutinin inhibited phage T4D adsorption to the acetone and ether-treated cells probably by blocking the phage-receptor sites on the cell wall. The blocking by concanavalin A and by wheat-germ agglutinin was reversed by alpha-methyl-D-glucoside and by alpha-methyl-N-acetyl-D-glucosaminide, respectively. Results suggested the possibility that coliphage T4D requires N-acetyl-D-glucosaminyl-glucose or glucosyl-D-glucosamine residues of the core of lipopolysaccharides for the initial attachment to the cell wall of Escherichia coli B.

Effect of plant lectins on gamma phage receptor sites of Bacillus anthracis.

Japanese Journal of MicrobiologyVolume 20, Issue 2 p. 147-149 ArticleFree Access Effect of Plant Lectins on γ Phage Receptor Sites of Bacillus anthracis Takashi Watanabe, Corresponding Author Takashi Watanabe Department of Microbiology, Mie University School of Medicine, MieRequests for reprints should be addressed to Dr. Takashi Watanabe, Department of Microbiology, Mie University School of Medicine, 2–174 Edobashi, Tsu-shi, Mie 514, Japan.Search for more papers by this authorToshiro Shiomi, Toshiro Shiomi Department of Microbiology, Mie University School of Medicine, MieSearch for more papers by this author Takashi Watanabe, Corresponding Author Takashi Watanabe Department of Microbiology, Mie University School of Medicine, MieRequests for reprints should be addressed to Dr. Takashi Watanabe, Department of Microbiology, Mie University School of Medicine, 2–174 Edobashi, Tsu-shi, Mie 514, Japan.Search for more papers by this authorToshiro Shiomi, Toshiro Shiomi Department of Microbiology, Mie University School of Medicine, MieSearch for more papers by this author First published: February 1976 https://doi.org/10.1111/j.1348-0421.1976.tb00921.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1Agrawal, B.B., and Goldstein, J.J. 1967. Protein-carbohydrate interaction. VI. Isolation of concanavalin A by specific adsorption on cross-linked dextran gels. Biochim. Biophys. Acta 147: 262– 271. 2Archibald, A.R., and Coapes, H.E. 1972. Blocking of bacteriophage receptor sites by concanavalin A. J. Gen. Microbiol. 73: 581– 584. 3Bartell, P.E., Orr, T.E., Reese, J.F., and Imaeda, T. 1971. Interaction of Pseudomonas aeruginosa strain BI. J. Virol. 8: 311– 317. 4Burger, M.M., and Goldberg, A.R. 1967. Identification of tumor-specific determinant on neoplastic cell surfaces. Proc. Nat. Acad. Sci. U.S. 57: 359– 366. 5Chatterjee, A.N. 1969. Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus. J. Bacteriol. 98: 519– 527. 6Glaser, L., Ionesco, H., and Schaeffer, P. 1966. Teichoic acids as components of a specific phage receptor in Bacillus subtilis. Biochim. Biophys. Acta 124: 415– 417. 7Jazwinski, S.M., Lindberg, A.A., and Kornberg, A. 1975. The lipopolysaccharide receptor for bacteriophages φX 174 and S 13. Virology 66: 268– 282. 8Lindberg, A.A. 1973. Bacteriophage receptors. Ann. Rev. Microbiol. 27: 205– 241. 9Nicolson, G.L., and Blaustein, J. 1972. The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces. Biochim. Biophys. Acta 266: 543– 547. 10Takeda, K., and Uetake, H. 1973. In-vitro interaction between phage and receptor lipopolysaccharide: a novel glycosidase associated with Salmonella phage ε15. Virology 52: 298– 314. 11Tamaki, S., and Sato, T. 1971. Role of lipopolysaccharides in antibiotic resistance and bacteriophage adsorption of Escherichia coli K-12. J. Bacteriol. 105: 968– 975. 12Vidaver, A.K., and Brock, T.D. 1966. Purification and properties of a bacteriophage receptor material from Streptococcus faeium. Biochim. Biophys. Acta 121: 298– 314. 13Watanabe, T. 1976. Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B. Canad. J. Microbiol. 22: (in press, No 5). 14Watanabe, T., Morimoto, A., and Shiomi, T. 1975. The fine structure and protein composition of γ phage of Bacillus anthracis. Canad. J. Microbiol. 21: 1889– 1892. 15Watanabe, T., and Shiomi, T. 1975. Inhibiting materials for γ phage adsorption to the cell wall of Bacillus anthracis, strain Pasteur No. 2-H. Japan. J. Microbiol. 19: 115– 121. 16Weider, W., and Primosigh, J. 1958. Biochemical paralleles between lysis by virulent phage and lysis by penicillin. J. Gen. Microbiol. 18: 513– 517. 17Yokokura, T. 1971. Phage receptor material in Lactobacillus casei cell wall. I. Effect of L-rhamnose on phage adsorption to the cell wall. Japan. J. Microbiol. 15: 457– 463. Citing Literature Volume20, Issue2February 1976Pages 147-149 ReferencesRelatedInformation

Effect of protein A on adsorption of bacteriophages to Staphylococcus aureus.

Experiments were performed to determine if protein A influenced the association of bacteriophages with Staphylococcus aureus. Bacteriophage adsorption was compared in a S. aureus strain rich in protein A and mutants of this strain with very little protein A, in a strain with little protein A, and in mutants of this strain with increased protein A. In addition, the effect of growth in mannitol-salt broth and trypsin digestion (known to reduce protein A) on bacteriophage absorption was measured. There was an inverse relationship between protein A content of strains and the quantity of bacteriophage absorbed. However, no inhibition of staphylococcal phages was obtained with purified soluble protein A. Protein A as a surface component rendered the bacteria more resistant to adsorption of staphylococcal typing phages presumably by masking the phage receptor sites. When protein A-deficient mutants were incubated with bacteriophages, there was survival of staphylococci with increased protein A content probably due to a selective action.

A pleiotropic mutation affecting expression of polar development events in Caulobacter crescentus.

A developmental mutant of C. crescentus with altered polar surface structures has been isolated. The mutant lacks a flagellum and pili, and may have an abnormal DNA phage receptor site. A revertant regains the normal structures simultaneously. This point mutation allows normal flagellin synthesis, stalk formation, equatorial cell division, and rate of growth. The mutant phenotype indicates that the assembly of the polar surface structures is coordinately regulated and independent of mechanisms regulating cell polarity and division.

Studies on the mechanism of phage adsorption: interaction between phage epsilon15 and its cellular receptor.

Abstract The temperate Salmonella phage ϵ15 and a soluble protein present at late times in ϵ15-infected cells can cleave the phage receptor site on the surface of sensitive cells. The phage receptor, a heteropolysaccharide containing mannose, rhamnose, and galactose is degraded by the specific cleavage of rhamnosylgalactose linkages. The products are a series of oligosaccharides. The soluble protein, purified from phage-infected cell lysates, has properties expected of a component of the phage tail. It adsorbs specifically to phage-sensitive cells, it interacts with anti-ϵ15 phage serum blocking its action on ϵ15 phage, and it contains a single polypeptide component which corresponds to one of the major polypeptides present in the ϵ15 virion by the criterion of SDS-acrylamide gel electrophoresis.

Intercellular transfer by phage receptor site lipopolysaccharide.

TREATMENT of Escherichia coli cells with lysozyme and EDTA partially removes the outer layer of the cell wall containing lipopolysaccharide (LPS), leaving osmotically unstable spheroplasts1. These can be infected with phage nucleic acid2 and can produce viable phage particles. Removal of LPS-containing phage receptor sites3–5, however, leaves spheroplasts resistant to infection by intact phages1. We now show that LPS, obtained from phage-sensitive cells by aqueous phenol extraction, can provide functional phage receptor sites to spheroplasts prepared from cells lacking receptor sites.

Transfer of Phage Receptor Sites

Transfer of Phage Receptor Sites

Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus.

A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.

Bacterial differentiation.

The foregoing studies are intended to define a differentiation process and to permit genetic access to the mechanisms that control this process. In order to elucidate the basic mechanisms whereby a cell dictates its own defined morphogenic changes, we have found it helpful to study an organism that can be manipulated both biochemically and genetically. We have attempted to develop the studies initiated by Poindexter,Stove and Stanier, and Schmidt and Stanier (16, 17, 20) with the Caulobacter genus so that these bacteria can serve as a model system for prokaryotic differentiation. The Caulobacter life cycle, defined in synchronously growing cultures, includes a sequential series of morphological changes that occur at specific times in the cycle and at specific locations in the cell. Six distinct cellular characteristics, which are peculiar to these bacteria, have been defined and include (i) the synthesis of a polar organelle which may be membranous (21-23), (ii) a satellite DNA in the stalked cell (26), (iii) pili to which RNA bacteriophage specifically adsorb (16, 33), (iv) a single polar flagellum(17), (v) a lipopolysaccharide phage receptor site (27), and (vi) new cell wall material at the flagellated pole of the cell giving rise to a stalk (19, 20). Cell division, essential for the viability of the organism, is dependent on the irreversible differentiation of a flagellated swarmer cell to a mature stalked cell. The specific features of the Caulobacter system which make it a system of choice for studies of the control of sequential events resulting in cellular differentiation can be summarized as follows. 1) Cell populations can be synchronized, and homogeneous populations at each stage in the differentiation cycle can thus be obtained. 2) A specific technique has been developed whereby the progress of the differentiation cycle can be accurately measured by adsorption of labeled RNA phage or penetration of labeled phage DNA into specific cell forms. This technique can be used to select for mutants blocked in the various stages of morphogenesis. 3) Temperature-sensitive mutants of Caulobacter that are restricted in macromolecular synthesis and development at elevated temperatures have been isolated. 4) Genetic exchange in the Calflobacter genus has been demonstrated and is now being defined. Two questions related to control processes can now readily be approached experimentally. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? The availability of temperature-sensitive mutants blocked at various stages of development permits access to both questions. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. Because the ultimate expression of cell changes are dependent on selective protein synthesis, specific messenger RNA production-either from DNA present in an organelle or from the chromosome-may prove to be a controlling factor in cell differentiation. We have begun studies with RNA polymerase purified from Caulobacter crescentus to determine whether cell factors or alterations in the enzyme structure serve to change the specificity of transcription during the cell cycle. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). The Caulobacter bacteria now present another system in which direct analysis of these control mechanisms is feasible.