SummaryMany species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K-12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC H ± H lacZ translational fusion was expressed at three-to fourfold higher levels in csrA wild-type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription-translation of flhDC H ± H lacZ in S-30 extracts and bound to the 5 H segment of flhDC mRNA in RNA mobility shift assays. The steady-state level of flhDC mRNA was higher and its half-life was < threefold greater in a csrA wild-type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post-transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.

The Bacillus cereus group comprises the four valid species Bacillus cereus, Bacillus mycoides, Bacillus fhuringiensis and Bacillus anthracis. Some isolates of B. cereus are known to be psychrotolerant (growth a t 7 O C or below). Here, specific sequence differences are described between the 165 rDNA, the 235 rDNA, the 165-235 rDNA spacer region and the genes of the major cold-shock protein homologue cspA in a variety of psychrotolerant and mesophilic B. cereus and B. mycoides strains. Randomly amplified polymorphic DNA analysis using three different primers clearly separated psychrotolerant strains of both species from the rest of the B. cereus group, as did inverse PCR patterns of the rDNA operons. These data strongly support a hitherto unrecognized fifth sub-group within the B. cereus species group comprising psychrotolerant, but not mesophilic, B. cereus strains. Despite the latter finding, the DNA sequences investigated exhibited a high degree of sequence similarity indicating a close relationship between the species of the B. cereus group. Considering the unusual importance of B. cereus in both food poisoning and food spoilage and to avoid merging all species of the group, a new species, Bacillus weihenstephanensis sp. nov., comprising psychrotolerant 'cereus' strains, is proposed. Isolates of the new species grow a t 4-7 "C but not a t 43 OC and can be identified rapidly using rDNA or cspA targeted PCR. The type strain is B. weihenstephanensis WSBC 10204T (= DSM 1182IT).

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.An important source of genome plasticity is derived from transpositional events of insertion sequence (IS) elements (34, 35). They generally encode no functions other than those involved in their mobility (for a review, see reference 30) and display a nonrandom distribution in the chromosome of Escherichia coli (9, 16). Many IS elements have been shown to activate the expression of neighboring genes, for example, through the formation of hybrid promoters or disruption of transcriptional repression. This has also been seen with cryptic operons, which depend upon mutations for activation. Two examples in E. coli are the bgl and ade operons, which can be activated by IS element insertion upstream or downstream of the promoter (18,42,49,50,53). The chitobiose operon, chb (formerly cel), was thought to be cryptic but can be induced by chitobiose, as well as being activated by IS element insertion upstream of the structural genes under noninducing conditions (40, 44).Flagellar motility enables bacteria to escape from detrimental conditions and to reach more favorable environments. In E. coli, the flagellar regulon involves the expression of at least 14 operons in a regulated cascade to produce functional flagellar and chemotaxis machinery (for a review, see reference 14). The flhD operon at the apex of the flagellar regulon has been identified as the primary target of regulation by many environmental factors (for a review, see reference 61). It consists of two genes, flhD and flhC, whose product...

SummaryWe used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüß and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.

We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::TnlO-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated A(nuoFGHIJK;L)-1. Cells (6) propose that E. coli might regulate the amount of energy recovered from NADH oxidation by modulating the relative levels of these two NADH dhs.The genetic loci nuo (NADH:ubiquinone oxidoreductase), which maps 51.5 and 51.8 min on the E. coli linkage map (33a), and ndh (NADH dehydrogenase), which maps at 25.1 min, encode NADH dhl and NADH dhII, respectively. Both loci were identified in related mutant strains, AN589 (52) and IY12 (6). Cells containing mutations in both nuo and ndh, e.g., those * Corresponding author. Phone: (708)