We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain 8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinoseregulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P R promoter. An arabinose-regulated c2 gene is present in the chromosome. 8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P R, driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic ␣-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with 8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.programmed cell lysis ͉ rPspA ͉ Rx1

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The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 X2831(pYA902). The C terminus consists of a serine-and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci.Understanding the biological role of extracellular polysaccharides produced by oral streptococci (Streptococcus mutans, S. sanguis, S. sobrinus, S. cnicetus, and S. rattus), herein designated mutans streptococci, in contributing to dental caries has significantly increased in the past several years (13,22,34). It has been suggested (10, 31) that these extracellular polysaccharides are mainly composed of two classes, namely, a-1,6-linked water-soluble glucan (dextran) and a-1,3-linked water-insoluble glucan (mutan). It has been shown that on the tooth surface, pellicle-associated dextrans and glucans are involved in the first step in dental caries, a sucrose-independent attachment of S. mutans to the tooth surface (27). Also, there is considerable evidence for the importance of proteins on the surface of S. mutans, such as surface protein antigen A (SpaA) (39), being involved in this sucrose-independent phase. The second step is sucrose dependent and involves adherence of bacteria in a much firmer bonding to the tooth surface and also the aggregation reaction between adjacent bacterial cells in different chains of streptococci. Both adherence and aggregation in the sucrose-dependent phase involve the synthesis of water-soluble and water-insoluble glucans under the control of cell-associated glucosyltransferase enzymes (34,43,78). Several lines of evidence also suggest the involvement of dextranases in the virulence of oral streptococci by playing some role in adherence and aggregation (32). These observations include the following: (i) dextranase (a-1,6-glucan-6-glucanohydrolase; EC 3.2.1.11) can partially degrade water-soluble glucan (7, 31); (ii) dextranase can inhibit the production of water-* Corresponding author. Mailing address: Washington University,