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The lower intestine of adult mammals is densely colonized with non-pathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (>14 days) onset of a long-lived (t1/2>16 weeks), highly specific anti-commensal IgA response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

To avoid costly biomass recovery in photosynthetic microbial biofuel production, we genetically modified cyanobacteria to produce and secrete fatty acids. Starting with introducing an acyl-acyl carrier protein thioesterase gene, we made six successive generations of genetic modifications of cyanobacterium Synechocystis sp. PCC6803 wild type (SD100). The fatty acid secretion yield was increased to 197 AE 14 mg∕L of culture in one improved strain at a cell density of 1.0 × 10 9 cells∕mL by adding codon-optimized thioesterase genes and weakening polar cell wall layers. Although these strains exhibited damaged cell membranes at low cell densities, they grew more rapidly at high cell densities in late exponential and stationary phase and exhibited less cell damage than cells in wild-type cultures. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production.

In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic ␣-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the ␤-lactamase signal sequence in the multicopy Asd ؉ pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd ؉ vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations ⌬crp-28 and ⌬asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10 9 CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.

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The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain 7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with 7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain 7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain 7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.Avian-pathogenic Escherichia coli (APEC) comprise a specific subset of pathogenic E. coli that cause extraintestinal diseases of poultry. Of the various forms of E. coli disease in poultry, the most common syndrome starts as a respiratory tract infection in 3-to 12-week-old broiler chickens and turkeys and frequently becomes more generalized. The air sacs are the first organs affected, and systemic spreading may result in pericarditis, perihepatitis, and an often fatal septicemia (15,29). APEC infections are frequently enhanced or initiated by predisposing factors, which include environmental conditions and viral or Mycoplasma infection (15, 29). O1, O2, and O78 are the most commonly encountered serogroups among APEC (15,29), and the majority of strains have been shown to belong to a limited number of clonal lineages (69, 70). APEC strains of high virulence are lethal for 1-day-old chicks when administered subcutaneously. Attributes associated with APEC strains include F1 (type 1) and P fimbrial adhesins (16, 21, 53, 66), resistance to serum and phagocytosis (21, 22, 52, 71), the aerobactin siderophore system (21, 41, 65), and colicin V (7, 23, 65, 71) (reviewed in references 15 and 29). Recently the tsh gene, encoding a temperature-sensitive hemagglutinin, first identified by Provence and Curtiss (54), was shown to be associated with APEC but not with E. coli isolated from the feces of healthy chickens (45).The tsh gene was first identified from APEC O78:K80 strain 7122 and, when cloned into E. coli K-12, was shown to impart mannose-resistant hemagglutination of chicken erythrocytes...

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