Evidence is presented that a sporulationessential or factor of Bacillus subtilis, a29, is synthesized as an inactive precursor (P3') and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the p31 amino terminus. A pulse-chase experiment demonstrated that r29 was derived from a preexisting protein, with appearance of radioactively labeled ar29 paralleling the disappearance of labeled P31. The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B. subtilis strain carrying a mutation in a locus (spollE) GBq) to 25 uCi/ml; then either the cultures were harvested or the label was "chased" by the addition of a 103-fold excess of unlabeled methionine with continued incubation prior to harvesting. Cell extracts were prepared as described (4) and treated overnight at 4°C with anti-o-29 monoclonal antibody (4) in a 0.5-ml reaction mixture containing 10 mM Tris (pH 7.8), 10 mM EDTA, 0.3 mg ofphenylmethylsulfonyl fluoride, 0.1% NaDodSO4, 0.2% deoxycholate, 1% Triton X-100, a concentrated extract from 10 ml of B. subtilis culture, and 20,ug of antibody. Extracts then were incubated in the same reaction buffer with rabbit anti-mouse immunoglobulin for 4 hr, followed by incubation with washed Staphylococcus aureus cells (11)

The Escherichia coli K-12 alpA gene product, when overproduced from a multicopy plasmid, leads to suppression of the capsule overproduction and UV sensitivity phenotypes of cells mutant

Evidence is presented that the sporulationessential locus spolIG codes for both 29 subtilis strain with a mutation at the spolIG locus (spoIIG41).The appearance of P25 and P21 occurs in this mutant at a time when P31 and Ca29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIlG41 strain with plasmid DNA carrying the structural gene for spolIG complements the Spo-phenotype and results in the synthesis of P31, a'29 p25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spolIG sequence encoded a protein that reacted with the anti-P31/Oa'29 monoclonal antibody and had the electrophoretic mobility of authentic P31.The spore-forming bacterium Bacillus subtilis synthesizes at least five forms of DNA-dependent RNA polymerase, which are distinguished by the promoter specificity determinant (o, factor) that each carries on a common core enzyme (1-5). One of the a-factors, cr29, is a protein (Mr, 29,000) that is detected only during endospore formation and was therefore predicted to be an important element of spore gene regulation (3, 6,

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon ؉ cells, increased 38-fold in lon ؉ cells carrying a Tn10::⌬kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::⌬kan insertion. Sequencing of both sides of the Tn10::⌬kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.Regulation of colanic acid capsular polysaccharide gene (cps) expression in Escherichia coli is multilayered, as evidenced by the numerous direct and indirect regulatory mechanisms that have been identified to date (for reviews, see references 6 and 8). The current model proposes two pathways for activating cps expression (6). One pathway involves a twocomponent regulatory system (24). RcsC (regulator of capsule synthesis), which has been described as a membrane-bound sensor protein based on homology with the sensor component of two-component sensor regulator pairs (24), appears to be activated by environmental stimuli, such as desiccation (18) or osmotic shock (22). Presumably, activated RcsC either directly or indirectly modifies RcsB, the proposed effector of the twocomponent system, which in turn activates cps expression (6, 7). In the alternate pathway, the other positive effector of cps expression, RcsA, presumably forms a complex with RcsB, resulting in the activation of cps expression (2, 6, 25). RcsA is highly unstable and appears to be degraded in a Lon-dependent fashion (26; for a review, see reference 6). In lon ϩ cells, RcsA levels are low, and these cells produce little colanic acid (26; for a review, see reference 6). Conversely, in ⌬lon cells, RcsA levels are high, leading to increased colanic acid production and mucoid colonies (26; for a review, see reference 6). In this model, both pathways require RcsB for high-level expression of cps (6).Current evidence suggests that additional regulators of cps expression exist: mutations in hns (6 min) (21, 29), capS (22.5 min) (14), opsX (62 min) (30), or capT (unknown location) (14) lead to an increase in colanic acid production. Furthermore, mutations within the rfa locus (82 min) both alter lipopolysaccharide structure and synthesis and increase colanic acid production (19). Given the complexity of the cps system, identification of additional cps regulators seems probable. (SG20780 [2]) strains carrying the zce-23::⌬kan insertion were assayed for ␤-galactosidase activity (Table 1). A 38-fold increase in the level of cps expression was observed in either Luria-Bertani (LB) or minimal (M63 salts, 0.4% glucose, 0.1% Casamino Acids) medium with the introduction of the zce-23:: ⌬kan insertion into a lon ϩ strain. The zce-23::⌬kan insertion had no effect on cps expression in ⌬lon cells.The increase in cpsB10::lacZ expression is abolished in lon ؉ s...

A phage-resistant mutant with a defect in a membrane component required for phage infections in Lactococcus lactis subsp. lactis C2 was transformed with a chromosomal library of the wild-ype, phagesensitive strain. Of the 4,200 transformants screened for phage sensitivity, three were positively identified as phage sensitive. A cause-and-effect relationship between the cloned chromosomal fragments and the phagesensitive phenotype was established on the basis of the following two criteria: (i) the frequency of loss of the cloned fragments in the absence of antibiotic selection pressure correlated with the frequency of loss of phage sensitivity; and (ii) phage sensitivity was transferred to 100% of recipient, phage-resistant cells transformed with the cloned fragment. The cloned chromosomal DNA from the three independent isolates was physically mapped with restriction endonucleases. The sizes of the cloned fragments were 9.6, 11.8, and 9.5 kb. Each fragment contained an identical stretch of DNA common to all three, which was 9.4 kb. The gene that conferred phage sensitivity was localized by subcloning to a 4.5-kb region. Further The specific adsorption of bacteriophages to host cell surface components has been well characterized in many bacteria. In Escherichia coli, for example, phages adsorb to oligosaccharides and proteins exposed on the outer surface of the host cell (40,54). Following adsorption, the mechanism of phage DNA translocation across the plasma membrane is less well understood, although host cell components required for this process have been identified (7,19), and a mechanism involving plasma membrane ion channels is likely in the case of phage T5 (5,20).Lactococci are gram-positive bacteria and have cell walls that are much thicker than those of gram-negative bacteria such as E. coli. Because of this, phages of lactococci and other gram-positive bacteria generally adsorb to the cell wall prior to interaction with the membrane (3, 28). However, it has been suggested that lactococcal phages may adsorb directly to the plasma membrane (32). This suggestion was based on an electron microscopic study of the surface of lactococci, which showed the plasma membrane protruding through holes in the cell wall (23), and the discovery of a phage-inactivating substance in the plasma membrane fraction of Lactococcus lactis subsp. lactis ML3 (32,33).Our laboratory has characterized phage receptors on the surface of two strains of L. lactis (44,46

A natural lactococcal isolate, Lactococcus lactis ssp. cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid). This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression. Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media. Ropy352 was examined for inducible lysogenic phages. No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction. The sugar compositions of the two phenotypically distinct exopolysaccharides were determined. The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively. The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively. Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.

Capsule (cps) gene expression in Escherichia coli is controlled by a complex network of regulators. Transcription of the cps operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of arcsA::lacZ transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation inlon or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA::lacZfusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression ofrcsA are independent of each other. A sequence motif, conserved between the E. coli cps promoter and theErwinia amylovora ams promoter and previously shown to be the RcsA-RcsB binding site, was identified in the rcsApromoter region and shown to be required for high-level expression ofrcsA.

We have previously found that plasmids carrying the Escherichia coli alp gene (now to be called alpA) suppress two phenotypes of a delta lon protease mutant, overproduction of capsular polysaccharide and sensitivity to UV light. Suppression of these lon phenotypes is most likely explained by the increased degradation of the Lon substrates responsible for these phenotypes. We have called this suppressing protease activity Alp protease. The Alp protease activity is detected in cells after introduction of plasmids carrying the alpA gene, which encodes an open reading frame of 70 amino acids. Insertions which abolish Alp activity interrupt this open reading frame. We have used Tn10 and lambda placMu mutagenesis to identify a chromosomal locus, slpA, that is required for alpA+ suppression of delta lon. This locus maps at 57 min, close to the chromosomal location of alpA. The expression of beta-galactosidase from a lac transcriptional fusion to slpA is increased six- to eightfold when the alpA+ gene is present on a multicopy plasmid. Therefore, AlpA acts as a transcriptional regulator of the slpA gene(s); activation of slpA transcription is necessary to suppress the phenotypes of a delta lon mutation. In an accompanying paper (J. E. Kirby, J. E. Trempy, and S. Gottesman, J. Bacteriol. 176:2068-2081, 1994), we show that neither AlpA nor SlpA is a component of the protease itself but that they are part of a regulatory cascade which leads to expression of the Alp protease.