The bacterial stringent response is triggered by deficiencies of available nutrients and other environmental stresses. It is mediated by 5'-triphosphate-guanosine-3'-diphosphate and 5'-diphosphate-guanosine-3'-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. Borrelia burgdorferi encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the B. burgdorferi stringent response is mediated by a single enzyme, RelBbu, with both (p)ppGpp synthase and hydrolase activities, and that a B. burgdorferi 297 relBbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the rel regulon in B. burgdorferi cultured at 34°C, and found that transcription of genes involved in glycerol metabolism is induced by rel Bbu. Culture of the wild type parental strain, the rel Bbu deletion mutant and its complemented derivative at 34°C and 25°C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the glp operon for glycerol uptake and metabolism in these three strains confirmed that rel Bbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures. These results confirm and extend previous findings regarding the stringent response in B. burgdorferi. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks.

The impressive disease spectrum of Streptococcus pyogenes (the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified the arc transcript as more abundant in stationary phase in addition to the sag and sda transcripts that had been previously identified. We found that in stationary phase, the stability of sagA, sda, and arcT transcripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded by pnpA]) is rate limiting for decay of sagA and sda transcripts in late exponential phase, since the stability of these mRNAs was greater in a pnpA mutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in a pnpA mutant, the sagA mRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.

SummaryThe paralogous ribonucleases J1 and J2, recently identified in Bacillus subtilis, have both endoribonucleolytic and 5Ј-to-3Ј exoribonucleolytic activities and participate in degradation and regulatory processing of mRNA. RNases J1 and J2 have partially overlapping target specificities, but only RNase J1 is essential for B. subtilis growth. Because mRNA decay is important in regulation of virulence factors of Streptococcus pyogenes (the group A streptococcus, GAS), we investigated the role of these newly described RNases in GAS. We found that conditional mutants for both RNases J1 and J2 require induction for growth, so we conclude that, unlike the case in B. subtilis, both of these RNases are essential for GAS growth, and therefore their functions are not redundant. We compared decay of representatives of the two classes of messages we had previously identified: Class I, which decay rapidly in exponential and stationary phase of growth (hasA and gyrA), and Class II, which are stable in stationary phase and exhibit a biphasic decay curve in exponential phase (sagA and sda). We report that RNases J1 and J2 affect the rate of decay of Class I messages and the length of the first phase in decay of Class II messages.

The global transcriptional regulator (p)ppGpp (guanosine-3-diphosphate-5-triphosphate and guanosine-3,5-bisphosphate, collectively) produced by the relA and spoT genes in Escherichia coli allows bacteria to adapt to different environmental stresses. The stringent response is a regulatory response that allows bacteria to adapt to a lack of nutrients and other environmental stresses (6). It causes accumulation of guanosine-3Ј-diphosphate-5Ј-triphosphate (pppGpp) and guanosine-3Ј,5Ј-bisphosphate (ppGpp), collectively referred to as (p)ppGpp or "magic spots." These nucleotides are synthesized by enzymatic phosphorylation of GDP and GTP to ppGpp and pppGpp, respectively, using ATP as a phosphate donor (6). In Escherichia coli, two different but highly homologous proteins are involved in (p)ppGpp synthesis: RelA, bound to ribosomes and activated by the presence of uncharged tRNA at the ribosomal A site which generally synthesizes (p)ppGpp in response to amino acid limitation (47), and SpoT, a cytosolic (p)ppGpp synthetase (15) which is responsive to changes in the availability of carbon, phosphate, and fatty acids as well as to changes in temperature and osmolarity (6, 34). SpoT is also a (p)ppGpp hydrolase (21, 31). Many gram-positive bacteria have only a single rel ortholog which exhibits both (p)ppGpp synthetase and hydrolase activity (29).(p)ppGpp acts as a global transcriptional regulator. The general effect of the stringent response is a decrease in rRNA, tRNA, and protein synthesis and a decrease in growth rate that results in bacterial adaptation to an environment scarce in nutrients (6). (p)ppGpp also influences many other bacterial physiological functions including competence (23), morphological and physiological differentiation and production of clavulanic acid and cephamycin C (25, 26), production of actinorhodin and undecylprodigiosin antibiotics (46), thermotolerance (48), adaptation to oxidative stress (30), and sensitivity to antibiotics. As regards the latter, E. coli strains able to synthesize (p)ppGpp in either a RelA-or a SpoT-dependent manner show a greater resistance to antimicrobials than strains that cannot produce (p)ppGpp (17). In E. coli, production of (p)ppGpp is also required for the accumulation of inorganic polyphosphate needed for degradation of proteins during starvation mediated by the Lon protease (28), and lack of inorganic polyphosphate synthesis in enteric bacteria is accompanied by a reduction in virulence (27).The relA and spoT gene products and (p)ppGpp mediate important aspects of virulence in a number of pathogens (8,20,34). For example, Vibrio cholerae relA is involved in the ability of V. cholerae to display pathogenicity in in vitro and in vivo models of infection (20), Mycobacterium tuberculosis rel-mediated adaptation to stationary phase is critical to long-term persistence of M. tuberculosis in mice (8), and mutations in Salmonella enterica serovar Typhimurium relA and spoT result in attenuation in animals (34). Not all rel mutants show decreased virulence. Mutation of ...

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel Bbu ) was produced by B31 as by N40 during in vitro growth (2 ؎ 0.5 and 4 ؎ 0.8 fg of rel Bbu mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel Bbu mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel Bbu mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel Bsu were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel Bbu promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel Bbu ATG start codon and identical 70 -like promoters were identified by primer extension and sequencing analysis. rel Bbu complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel Bbu mRNA. These results indicate that rel Bbu is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.

Genes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). We report that in GAS, stk is required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of ␣-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that the stk deletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.Borrelia burgdorferi, the cause of Lyme disease, modulates its gene expression in the presence of host cells. As an example of this modulation, the relative expression of the ospA and ospC genes is altered as B. burgdorferi passes from a tick to a mammalian host (32); a similar down-modulation-up-modulation of OspA and OspC is observed during coculture of B. burgdorferi with tick cells in vitro (27). This oscillation in gene expression may be mediated by a regulatory loop involving changes in the expression of 54 and S , suggesting that B. burgdorferi growing in the presence of tick cells may undergo a series of as-yet-uncharacterized global regulatory changes (36). It also suggests that study of the reciprocal interactions between B. burgdorferi and tick cells could illuminate aspects of gene modulation in B. burgdorferi and the genes and gene products involved (17,36).Flexible adaptation by bacterial pathogens to rapidly changing host environments requires the interplay of regulators generated in response to signals triggered by environmental stimuli. One such adaptive response is the stringent response, a global response triggered by nutritional stress when protein synthesis is blocked by amino acid starvation (10, 13, 34). The stringent response causes up-regulation of protein degradation and amino acid synthesis, down-regulation of nucleic acid and protein synthesis, and alteration of many cellular functions, including transcription, translation, replication, transport, and metabolism, and it is accompanied by slow growth (10, 34). In Escherichia coli, the stringent response involves RelA, a synthase, and SpoT, a hydrolase, which synthesize and degrade, respectively, ppGpp and pppGpp [(p)ppGpp], the alarmon (master regulator, second message) that is the proximate mediator of this response (9, 10). In some bacterial pathogens, such as Mycobacterium t...

SummaryThe chromosomal paralogous gene family 36 encodes for four lipoproteins with high amino acid homology that are expressed in vivo in humans and animals and are immunogenic. Transcriptional analysis of the bmp gene cluster indicated that all four genes of this cluster are expressed in vitro and constitute two transcriptional units with a complex pattern of transcription, including alternative monocistronic and polycistronic messages. One unit consists of bmpD, whose transcription is coupled to the transcription of the ribosomal protein genes, rpsG and rpsL. The second unit includes bmpC, bmpA and bmpB. The simultaneous expression of the four bmp genes in Borrelia burgdorferi suggests that their gene products may have either different or complementary functions. Primer extension experiments identified promoters for bmpD, bmpC and bmpA, but not for bmpB. The concentration of genespecific mRNA paralleled its promoter homology to the Escherichia coli s 70 promoter. The linkage of bmpD expression to rpsL and rpsG suggests that the expression of this gene may be controlled by growthrelated global regulation mechanisms in B. burgdorferi. These results indicate that the bmp family constitutes a good model for the investigation of complex regulation of chromosomal gene expression in this bacteria.