- Email: [email protected]
Initiation of bacterial development
170
Initiation of bacterial development James A Hoch The discovery of a mitotic apparatus in bacteria has led to significant recent progress being made in understanding the regulatory connections between the cell cycle, chromosome segregation and the onset of developmental processes in sporulation. The control of developmental transcription by antagonism between protein kinase and protein phosphatase reached a new level of complexity with the discovery of peptide inhibitors of protein phosphatases that cycle between the interior and exterior cell surface as information messengers. New mechanisms of developmental regulation are being uncovered in a variety of microbial systems.
Addresses The Scripps Research Institute, Molecular and ExperimentalMedicine, Division of Cellular Biology, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 9203?; USA; e-mail: [email protected]
Current Opinion in Microbiology 1998, 1:170-174 http://biomednet.com/elecref/1369527400100170 © Current Biology Ltd ISSN 1369-5274
Abbreviation GFP
green fluorescent protein
Introduction One of the more interesting features of the lifestyle of certain bacteria and lower eukaryotes is the formation of differentiated structures containing environmentally resistant spores. The initiation of this developmental process requires the coordinated functioning of complex signal transduction networks that allow a variety of signal inputs from metabolism, the environment, and the cell cycle to impact the decision to grow or differentiate. The mechanisms by which these signals are recognized and regulate gene expression arc being uncovered in several systems. The nature of the signals that affect the activity of these mechanisms remains a mystery. In bacterial development, on which this review will focus, important advances have been made in understanding the interaction between the cell cycle and development. Once development begins, fascinating regulatou interactions between cell compartments unfold that have been best described in a recent review [1]. C o o r d i n a t i o n of t h e cell cycle and development In sporulating bacilli, development begins when one chromosome of a cell is segregated into a forespore compartment with a second chromosome remaining in the mother cell. The segregation into the forespore differs from normal cell division in that the sporulation septum is formed with only 30% of the chromosome in the forespore compartment. The remaining 70% in the mother cell is translocated to the forespore by a conjugation-like
mechanism involving the product of the spolIIE locus [2]. In verification of their translocation mechanism, Wu and Errington [3] have shown that the SpoIIIE protein is associated with the cellular membrane and located near the center of the sporulation septum. This unusual mechanism for chromosome segregation into the forespore compartments may ensure that different signals between chromosome replication and septation are used in cellular division and sporulation. What the signals are remains a myste~. Much activity in the past year in Bacillus subtilis has centered around an obscure Stage 0 sporulation locus, spoOJ, identified during the course of gcnome sequencing [4] as homologous to the parA-parB genes of plasmids. This homology suggested that the proteins encoded by this locus may have a role in chromosome segregation and that chromosome segregation may be different in division and sporulation. Subsequent studies showed that the parB homolog of the spoOJ locus was required to specify the orientation and directionality of transfer of the chromosome through the sporulation septum, suggesting that this protein was part of a centromere-like apparatus anchoring the origin to the pole of the cell [5"]. An elegant series of experiments by Webb et aL [6"'] established that the origin region of the chromosome is located at the ends of the cells during sporulation and preferentially towards the pole of the cells during division These investigators labeled the chromosome origin with tandem copies of the Escherichia colt /ac operator and visualized its position in the cell using a LacI repressor fused to green fluorescent protein (GFP). These studies were highly suggestive that chromosome segregation was accomplished via a mitotic-like apparatus. At the same time, an investigation into the function of the parA-parB locus of Caulobacter crescentus revealed that ParB binds to the origin of replication and that both ParA and ParB localized to the poles of cells [7"]. These data suggested that the Par proteins are likely to be involved with partitioning newly replicated chromosomes to the poles of the cell and could function as components of a mitotic-like apparatus. Similar studies of the Spo0J protein, which is homologous to ParB, showed that a Spo0J-GFP bound to the origin region of the chromosome and was located to the poles at the cell, suggesting that a mitotic-like apparatus was responsible for chromosome partitioning [8"',9",10]. These independent studies establish that a mitotic-like apparatus exists in bacteria and that ParB and its homologs are associated with the apparatus in B. subtilis and may be essential for its functioning in C. oescentus. The role of the ParA homologs in both organisms is now obscure. ParA is an essential protein in C. crescentus and is
Initiation of bacterial development Hoch
located at the poles similarly to ParB [7"°]. In B. subtilis, the ParA homolog is responsible for the sporulation defect of mutants in the ParB homolog Spo0J [11]. The mechanism responsible for this phenotypc remains a mystery. Recent studies have implicated D N A primase in chromosome partitioning in E. co/i because certain dnaG mutants arc defective in chromosome segregation but not in DNA synthesis [12]; furthermore, suppressor studies designed to identity proteins that interact with DNA primase and the segregation mechanism revealed mutations in a variety of genes [13]. Whether DNA primase and other proteins are directly associated with the mitotic apparatus or not has yet to be determined. T h e fact that both phosphorylation and proteolysis are now known to be involved in C. crescentus cell-cycle control, as in eukaryotes, indicates that cell-cycle regulation and its relationship with mitosis will be complex and involve many proteins [14"']. There is a great deal more to be learned about this mitotic-like apparatus and how it communicates with elements regulating development in B. subti/is and (7,. crescentus [15"'].
Protein aspartate phosphatases in phosphorelay control T h e initiation of developmental transcription in B. subtilis sporulation is dependent on phosphorylated Spo0A transcription factor. Phosphorylation is the end result of signal transduction through the phosphorelay consisting of two kinases, KinA and KinB, which phosphorylate the SpoOF response regulator, and a phosphotransferase, Spo0B, which transfers the phosphate from Spo0F-P to the Spo0A transcription factor [16]. It was originally postulated that the phosphorelay was a signal integration mechanism allowing a variety of positive and negative regulators to act on the various components to affect the decision to divide or sporulate [16,17]. Several lines of evidence are consistent with this hypothesis. One of the more intriguing mechanisms regulating the phosphorelay is inhibition by the protein aspartate phosphatases, RapA and RapB, which dephosphorylate SpoOF and thereby prevent the formation of active Spo0A transcription factor [18]. RapA phosphatase activity was found to be regulated by two mechanisms [19"]: transcription of the rapA gene was controlled and, as part of the same transcript, a small protein of 44 amino acids was produced. Genetic studies showed that this protein, PhrA, was a regulator of Rap phosphatase activity. Examination of the primary sequence of the peptide predicted that a signal peptidase cleaved the protein resulting in the secretion of the 20 amino acid carboxyl portion. Complementation of a phrA mutant by synthetic peptides revealed that the last six carboxy-terminal residues are sufficient to regulate the phosphatase [19"]. These studies also showed that the sporulation deficiency of oligopeptide transport mutants resulted from their inability to transport peptides to inhibit RapA and RapB.
171
T h e mechanism by which these peptides regulate RapA phosphatases was theorized to be the direct inhibition of enzymatic activity and recent experiments have confirmed this idea. Perego [20"] showed that a peptide corresponding to the last five amino acids of PhrA was an inhibitor of the dephosphorylation of SpoOF by RapA and any single amino acid change in this pentapeptide resulted in an inactive peptide. Furthermore, the PhrA pentapeptide did not inhibit RapB, showing that the peptides arc specific and this specificity is encoded in the amino acid sequence. That the peptide works by being internalized was also shown by inducing the PhrA pentapeptide from a constructed expression vector [21"]. T h e question remains as to why the phosphatases are regulated by peptides that are processed from exported proteins. Originally; it was believed that the peptides may be a means for cell-cell communication [19"]. Perego [20"] has recently suggested that cell-cell communication is an unlikely scenario, envisioning instead that export and processing of the 44 amino acid Phr proteins are checkpoints for some process occurring in the periplasm and, once this is completed, the peptides can be processed and imported. The peptides may be a communication link from the periplasm to the cell interior.
A novel kinase inhibitor of sporulation Sensor kinases of signal transduction systems are believed to sense something to activate their functions. Understanding what is recognized by the sensor kinases for sporulation would be a big step forward in understanding development. Recently Wang et al. [22"] described a protein inhibitor of the primary sporulation kinase KinA that was encoded in an operon of genes of unknown function but regulated by nitrogen availability. This anti-kinase inhibitor, KipI, inhibited the autophosphorylation of KinA but not the phosphotransferase reaction. Another gene of the operon codes for an anti-anti-kinase protein, KipR, which neutralizes KipI activity. Thus, KinA inhibition by this system is regulated by the transcription of the kip gene operon and by the dynamics of the interaction between KipI and KipR. Surprisingly, it was found that KipI interacts with the catalytic domain of KinA, specifically the ATP-binding site, not the sensor domain. This is the first example of two-component signal transduction inhibitors that act on the catalytic domain of sensor kinases to counteract the signals activating the sensor domain of these kinases.
Overview of sporulation initiation in B. subtilis It is now clear that the phosphorelay signal transduction pathway provides many access points for regulation by inhibitors induced by cellular processes presumably antithetical to sporulation (Figure 1). This includes at least two phosphatases for Spo0F-P regulated by different inducers and a phosphatase for Spo0A-P. T h e cffectors of KinA and KinB are unknown but is now known that
172
Cell regulation
KinB must be sensing something in the periplasm [23[. Thus, we now have an appreciation of the mechanisms that regulate the level of Spo0A-P but the cellular functions and molecules that regulate the mechanisms remain obscure.
Sensor kinases in development Development into spores in Al),xococcus xanthus is a communal affair in which 105 cells form an organized mound or fruiting body inside which the spores are formed. Although ,.11. xanthus is Gram negative, it shares with B. subti/is and lower eukaryotes the induction of sporulation by nutrient limitation. In ,.11. xanthus the
sasS gene codes for a transmembrane sensor histidine kinase that regulates early developmental gcne expression [24]. It is thought that this kinase may phosphorvlate a NtrC-like regulator that activates transcription of sigma-54 promoters known to be expressed during early development [13]. A gene, fruA, has been identified that encodes a transcriptional regulator required for sporulation and normal fruiting body morphogenesis [25]. FruA appears to be structurally similar to response regulators of two-component systems and may be activated by phosphorylation. Typical two-component histidine kinases have been found to be required for preventing gene expression and sporulation in Dico,ostelium [26",27"] and
Figure 1
Cell membrane
Cytoplasm KipR
KinA ATP
ADP
<~
'
Kipl , ~
l-R [Inactive]
KinA-~) KapB KinB-(~
) Import
O F - ( ~ ~
OB-(~ %"-&'~"--'~ OA-(~
~
PhrA
RapA
L
L.~)
r
spollG
|l
I
PhrA
Current Opinion in Microbiology
Regulatory interactions in the phosphorelay controlling sporulation in B. subtilis. Phosphorylated OA is a transcription activator of many genes, including spollG. Its phosphorylation level and activity are subject to dephosphorylation by OE phosphatase and by RapA and RapB phosphatases that dephosphorylate OF-P. The RapA phosphatase is regulated by a peptide product of the phrA locus processed after export to the exterior of the cell. Phosphate input by KinA is regulated by an anti-kinase, Kipl, and an anti-anti-kinase, KipR. KinB is located in the cellular membrane and its activity is controlled by KapB. Circled P, phosphorylation.
Initiation of bacterial development Hoch
conidiophore development in Neurospora crassa [28,29"]. Thus, signal transduction by two-component systems is likely to play a major role in environmental sensing and development in a variety of bacteria and sporulating fungi.
173
the mechanisms are exceedingly complex and precisely coordinated and we have really only had a glimpse of this machinery.
Acknowledgements GTPases in development Era and Obg GTP-binding proteins with GTPase activity are enigmatic components of bacterial cells thought to have roles in signal transduction because that is the role of similar proteins in higher organisms [30]. No one has defined what these proteins do in bacteria. In B. subtilis, the Obg protein is essential for growth and the initiation of sporulation but not for later stages of sporulation [31]. An Ohg homolog has been identified in Streptomyces species and increasing the cellular content of Obg from a multicopy plasmid repressed aerial mycetium development [32]. What pathway or process is being affected by these proteins? A low molecular weight GTPase, MglA, similar to the Ras/Rab/Rho superfamily required for multicelhdar development and gliding mobility has been found in ,.11. xanthus [33"']. Surprisingl'> the yeast GTPase Sarlp is able to complement the sporulation defect of an mg/A deletion. An undefined second site mutation, rpm, allowed Sarlp to suppress both developmental and motility defects of the deletion strain. Because the role of Sarlp is also unknown, the functions of MglA in motility and development remain murky.
Butyrolactone-regulating factors in
$treptomyces development Streptomyces species produce a family of butyrolactone autoregulators that control differentiation and secondary metabolism. T h e regulators can be classified into three types on the basis of structural differences. Receptors for each class, A, IM-2 and VG, have now been cloned and characterized [34]. These receptors have homology to the TetR family of transcriptional repressors and are specific for each butyrolactone. T h e A-factor butyrolactone is essential for aerial mycetium formation and streptomycin production in Streptomyces gHseus [351. This phenotype of an A-factor mutant may be reversed by the introduction of a Ser/Thr kinase gene from Streptomt'ces coe/icoloz, suggesting that the kinase is part of the A-factor regulatory network [35].
Conclusions T h e only feature that all microbial development seems to have in common is that induction of the process occurs under conditions of nutrient deprivation or high cell density. Many of the organisms appear to place some induction control on molecules that may signal cell density such as A-factor or butyrolactones, what the organism had in mind for this type of regulation can only be guessed at. At this point, most microbial development is still at t h e stage o f d e f i n i n g m e c h a n i s m s o f control r a t h e r t h a n w h a t is c o n t r o l l i n g t h e m e c h a n i s m : h o w e v e r , it is clear t h a t
The author wnuld like to acknowledge the advice of Heidi Kaplan and Kcith Chatcr in idcntit'ying papers of interests.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ,, of outstanding interest 1.
Stragier P, Losick R: Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 1996, 30:297-341.
2.
Wu U, Lewis PJ, AIImansberger R, Hauser PM, Errington J: A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev 1995, 9:1316-1326.
3.
Wu U, Errington J: Septal localization of the SpolllE chromosome partitioning protein in Bacillus subtilis. EMBO J 1997, 16:2161-2169.
4.
Ogasawara N, Yoshikawa H: Genes and their organization in the replication origin region of the bacterial chromosome. Mol Microbiol 1992, 6:629-634.
5. •
Sharpe ME, Errington J: The Bacillus subtilis soj-spoOJ locus is required for a centromere-like function involved in prespore chromosome partitioning. Mo/Microbio/1996, 21:501-509. This work presages the role of the spo0J proteins in mitosis. 6. o•
Webb CD, Telernan A, Gordon S, Straight A, Belmont A, Lin DC, Grossman AD, Wright A, Losick R: Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subti/is. Cell 1997, 88:667-674. This article describes a very clever method for labelling the origin of chromosome replication with /ac operators and it demonstrates the behavior of origins in mitosis. 7. •,
Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Ceil 1997, 88:675-684. An excellent demonstration of the functions of ParA and ParB in chromosome segregation and mitosis. ParB was found to bind to sequences adjacent to the origin of replication and this region was tethered to the poles of the cell during a specific time of the cell cycle. ParA also segregated to the poles suggesting it was part of the mitotic appartus. 8. *•
Glaser P, Sharpe ME, Raether B, Perego M, Ohlsen KL, Errington J: Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev 1997, 11:1160-1168. The Spo0J protein of Bacillus subtih's is equivalent to the ParB of Caulobacter crescentus and was shown to localize close 1o the cell poles using immunofluorescence and a GTP fusion to Spo0J. These loci replicate in concert with the initiation of new rounds of DNA replication and they function to position the oriC region of the chromosome providing evidence for a mitotic-like apparatus. 9. •
Lin DCH, Levin PA, Grossman AD: Bipolar localization of a chromosome partition protein in Bacillus subtilis. Proc Nat/ Acad Sci USA 1997, 94:4721-4726. An independent study showing the segregation of Spo0J proteins. See annotation [8*']. 10.
Lewis PJ, Errington J: Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and colocalization with the Spo0J partitioning protein. Mo/Microbiol 1997, 25:945-954.
11.
Ireton K, Gunther NW, Grossman AD: spoOJ is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subti/is. J Bacterio/1994, 76:5320-532g.
12.
Versalovic J, Lupski JR: Missense mutations in the 3' end of the Escherichia colt dnaG gene do not abolish primase activity but do confer the chromosome-segregation-defective (par) phenotype. Microbiology 1994, 143:585-594.
13.
Keseler IM, Kaiser D: Sigma 54, a vital protein for Myxococcus xanthus. Proc Natl Acad Sci USA 1997, 94:1979-1984.
1 ?4
Cell regulation
14. 6.
Domian IJ, Quon KC, Shapiro L: Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the Gl-to-S transition in a bacterial cell cycle. Ceil 1997, 90:415-424. This article demonstrates the regulatory modifications of an essential transcription regulator for the cell-cycle and suggests that cell cycle regulation in bacteria may be similar to that in eukaryotes. This may be what everyone is researching in 1998. 15. Wheeler RT, Shapiro L: Bacterial chromosome segregation: is e° there a mitotic apparatus? Cell 1997, 88:577-579. An insightful review of mitosis in bacteria and of references [6"*,7"']. 16.
Burbulys D, Trach KA, Hoch JA: The initiation of sporulation in Bacillus subtilis is controlled by a multicomponent phosphorelay. Ceil 1991, 64:545-552.
17.
Ohlsen KL, Grimsley JK, Hoch JA: Deactivation of the sporulation transcription factor Spo0A by the SpoOE protein phosphatase. Proc Nat/Acad Sci USA 1994, 91:1756-1760.
18.
Perego M, Hanstein CG, Welsh KM, Djavakhishvili T, Glaser P, Hoch JA: Multiple protein aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in Bacillus subtilis. Ceil 1994, 79:1047-1055.
19. ••
Perego M, Hoch JA: Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc Nat/Acad Sci USA 1996, 93:1549-1553. This reseach showed that a phosphatase regulating development was controlled by a peptide which is first secreted and subsequently reimported by the oligopeptide permease. These studies show that the peptide functioned by inhibition of phosphatase activity not by regulation of phosphatase gene expression. 20. ••
Perego M: A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay. Proc Nat/Acad Sci USA 1997, 94:8612-8617. A direct demonstration of the in vitro inhibition of protein aspartate phosphatases by regulatory peptides and an exploration of the specificity determinants. This work forms the basis for the hypothesis that peptides communicate the status of extramembranous events to the cell. Lazazzera BA, Solomon JM, Grossman AD: An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell 1997, 89:917-925. This paper demonstrates that producing the mature peptides that regulate phosphatases inside the cell obviates the need for secretion and importation. These studies confirm that the oligopeptide permease is responsible for import of the peptides.
24.
Yang C, Kaplan HB: Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression. J Bacterio/1997, 179:7759-7767
25.
Ogawa M, Fujitani S, Mao X, Inouye S, Komano T: FruA, a putative transcription factor essential for the development of Myxococcus xanthus. Mo/ Microbio/1996, 22:757-767
26. .
Wang N, Shaulsky G, Escalante R, Loomis WF: A twocomponent histidine kinase gene that functions in Dictyostelium developmenL EMBQ J 1996, 15:3890-3898. A two-component kinase has been identified in Dictyostefium development, proof that it is not just for B. subti/is development anymore. See also [27"]. 27. •
Schuster SS, Noegel AA, Oehme F, Gerisch G, Simon MI: The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium. EMBO J 1996, 15:3880-3889. See annotation [26°]. 28.
29. •
Alex LA, Borkovich KA, Simon Ml: Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase. Proc Nat/ Acad Sci USA 1996, 93:3416-3421. A two-component kinase in the hyphal development of fungi: where will it end? Agene coding for a histidine kinase with a response regulator domain was found to be involved in osmoregulation and hyphal development. Deletion of the gene for this kinase results in aberrant hyphal structure that is enhanced under conditions of osmostress. 30.
Pillutla RC, Ahnn J, Inouye M: Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coil, causes a dominant-negative phenotype. FEMS Microbiol Lett 1996, 143:47-55.
31.
Kok J, Trach KA, Hoch JA: Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg. J Bacteriol 1994, 176:7155-7160.
32.
Okamoto S, Itoh M, Ochi M: Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation. J Bacteriol 1997, 179:170-179.
21. •
22. •-
Wang L, Grau R, Perego M, Hoch JA: A novel histidine kinase inhibitor regulating development in Bacillus subtilis. Genes Dev 199"7, 11:2569-2579. A demonstration of a new type of two-component kinase inhibitor targeted to the catalytic domain and its regulation by an anti-anti-kinase. 23.
Dartois V, Liu J, Hoch JA: Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis. Mo/ Microbio/1997, 25:39-51.
Schumacher MM, Enderlin CS, Selitrennikoff CP: The osmotic1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast. Curr Microbio/1997, 34:340-347.
33. •.
Hartzell PL: Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase. Microbiology 1997, 94:9881-9686. A fascinating demonstration of the complementation of a M. xanthus GTPase with a yeast counterpart. 34.
Waki M, Nihira T, Yamada Y: Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Strepfomyces sp. strain FRI-5. J Bacteriol 1997, 179:5131-5137.
35.
Ueda K, Umeyama T, Beppu T, Horineuchi S: The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2). Gene 1996, 169:91-95.
Initiation of bacterial development James A Hoch The discovery of a mitotic apparatus in bacteria has led to significant recent progress being made in understanding the regulatory connections between the cell cycle, chromosome segregation and the onset of developmental processes in sporulation. The control of developmental transcription by antagonism between protein kinase and protein phosphatase reached a new level of complexity with the discovery of peptide inhibitors of protein phosphatases that cycle between the interior and exterior cell surface as information messengers. New mechanisms of developmental regulation are being uncovered in a variety of microbial systems.
Addresses The Scripps Research Institute, Molecular and ExperimentalMedicine, Division of Cellular Biology, 10550 North Torrey Pines Road, NX-1, La Jolla, CA 9203?; USA; e-mail: [email protected]
Current Opinion in Microbiology 1998, 1:170-174 http://biomednet.com/elecref/1369527400100170 © Current Biology Ltd ISSN 1369-5274
Abbreviation GFP
green fluorescent protein
Introduction One of the more interesting features of the lifestyle of certain bacteria and lower eukaryotes is the formation of differentiated structures containing environmentally resistant spores. The initiation of this developmental process requires the coordinated functioning of complex signal transduction networks that allow a variety of signal inputs from metabolism, the environment, and the cell cycle to impact the decision to grow or differentiate. The mechanisms by which these signals are recognized and regulate gene expression arc being uncovered in several systems. The nature of the signals that affect the activity of these mechanisms remains a mystery. In bacterial development, on which this review will focus, important advances have been made in understanding the interaction between the cell cycle and development. Once development begins, fascinating regulatou interactions between cell compartments unfold that have been best described in a recent review [1]. C o o r d i n a t i o n of t h e cell cycle and development In sporulating bacilli, development begins when one chromosome of a cell is segregated into a forespore compartment with a second chromosome remaining in the mother cell. The segregation into the forespore differs from normal cell division in that the sporulation septum is formed with only 30% of the chromosome in the forespore compartment. The remaining 70% in the mother cell is translocated to the forespore by a conjugation-like
mechanism involving the product of the spolIIE locus [2]. In verification of their translocation mechanism, Wu and Errington [3] have shown that the SpoIIIE protein is associated with the cellular membrane and located near the center of the sporulation septum. This unusual mechanism for chromosome segregation into the forespore compartments may ensure that different signals between chromosome replication and septation are used in cellular division and sporulation. What the signals are remains a myste~. Much activity in the past year in Bacillus subtilis has centered around an obscure Stage 0 sporulation locus, spoOJ, identified during the course of gcnome sequencing [4] as homologous to the parA-parB genes of plasmids. This homology suggested that the proteins encoded by this locus may have a role in chromosome segregation and that chromosome segregation may be different in division and sporulation. Subsequent studies showed that the parB homolog of the spoOJ locus was required to specify the orientation and directionality of transfer of the chromosome through the sporulation septum, suggesting that this protein was part of a centromere-like apparatus anchoring the origin to the pole of the cell [5"]. An elegant series of experiments by Webb et aL [6"'] established that the origin region of the chromosome is located at the ends of the cells during sporulation and preferentially towards the pole of the cells during division These investigators labeled the chromosome origin with tandem copies of the Escherichia colt /ac operator and visualized its position in the cell using a LacI repressor fused to green fluorescent protein (GFP). These studies were highly suggestive that chromosome segregation was accomplished via a mitotic-like apparatus. At the same time, an investigation into the function of the parA-parB locus of Caulobacter crescentus revealed that ParB binds to the origin of replication and that both ParA and ParB localized to the poles of cells [7"]. These data suggested that the Par proteins are likely to be involved with partitioning newly replicated chromosomes to the poles of the cell and could function as components of a mitotic-like apparatus. Similar studies of the Spo0J protein, which is homologous to ParB, showed that a Spo0J-GFP bound to the origin region of the chromosome and was located to the poles at the cell, suggesting that a mitotic-like apparatus was responsible for chromosome partitioning [8"',9",10]. These independent studies establish that a mitotic-like apparatus exists in bacteria and that ParB and its homologs are associated with the apparatus in B. subtilis and may be essential for its functioning in C. oescentus. The role of the ParA homologs in both organisms is now obscure. ParA is an essential protein in C. crescentus and is
Initiation of bacterial development Hoch
located at the poles similarly to ParB [7"°]. In B. subtilis, the ParA homolog is responsible for the sporulation defect of mutants in the ParB homolog Spo0J [11]. The mechanism responsible for this phenotypc remains a mystery. Recent studies have implicated D N A primase in chromosome partitioning in E. co/i because certain dnaG mutants arc defective in chromosome segregation but not in DNA synthesis [12]; furthermore, suppressor studies designed to identity proteins that interact with DNA primase and the segregation mechanism revealed mutations in a variety of genes [13]. Whether DNA primase and other proteins are directly associated with the mitotic apparatus or not has yet to be determined. T h e fact that both phosphorylation and proteolysis are now known to be involved in C. crescentus cell-cycle control, as in eukaryotes, indicates that cell-cycle regulation and its relationship with mitosis will be complex and involve many proteins [14"']. There is a great deal more to be learned about this mitotic-like apparatus and how it communicates with elements regulating development in B. subti/is and (7,. crescentus [15"'].
Protein aspartate phosphatases in phosphorelay control T h e initiation of developmental transcription in B. subtilis sporulation is dependent on phosphorylated Spo0A transcription factor. Phosphorylation is the end result of signal transduction through the phosphorelay consisting of two kinases, KinA and KinB, which phosphorylate the SpoOF response regulator, and a phosphotransferase, Spo0B, which transfers the phosphate from Spo0F-P to the Spo0A transcription factor [16]. It was originally postulated that the phosphorelay was a signal integration mechanism allowing a variety of positive and negative regulators to act on the various components to affect the decision to divide or sporulate [16,17]. Several lines of evidence are consistent with this hypothesis. One of the more intriguing mechanisms regulating the phosphorelay is inhibition by the protein aspartate phosphatases, RapA and RapB, which dephosphorylate SpoOF and thereby prevent the formation of active Spo0A transcription factor [18]. RapA phosphatase activity was found to be regulated by two mechanisms [19"]: transcription of the rapA gene was controlled and, as part of the same transcript, a small protein of 44 amino acids was produced. Genetic studies showed that this protein, PhrA, was a regulator of Rap phosphatase activity. Examination of the primary sequence of the peptide predicted that a signal peptidase cleaved the protein resulting in the secretion of the 20 amino acid carboxyl portion. Complementation of a phrA mutant by synthetic peptides revealed that the last six carboxy-terminal residues are sufficient to regulate the phosphatase [19"]. These studies also showed that the sporulation deficiency of oligopeptide transport mutants resulted from their inability to transport peptides to inhibit RapA and RapB.
171
T h e mechanism by which these peptides regulate RapA phosphatases was theorized to be the direct inhibition of enzymatic activity and recent experiments have confirmed this idea. Perego [20"] showed that a peptide corresponding to the last five amino acids of PhrA was an inhibitor of the dephosphorylation of SpoOF by RapA and any single amino acid change in this pentapeptide resulted in an inactive peptide. Furthermore, the PhrA pentapeptide did not inhibit RapB, showing that the peptides arc specific and this specificity is encoded in the amino acid sequence. That the peptide works by being internalized was also shown by inducing the PhrA pentapeptide from a constructed expression vector [21"]. T h e question remains as to why the phosphatases are regulated by peptides that are processed from exported proteins. Originally; it was believed that the peptides may be a means for cell-cell communication [19"]. Perego [20"] has recently suggested that cell-cell communication is an unlikely scenario, envisioning instead that export and processing of the 44 amino acid Phr proteins are checkpoints for some process occurring in the periplasm and, once this is completed, the peptides can be processed and imported. The peptides may be a communication link from the periplasm to the cell interior.
A novel kinase inhibitor of sporulation Sensor kinases of signal transduction systems are believed to sense something to activate their functions. Understanding what is recognized by the sensor kinases for sporulation would be a big step forward in understanding development. Recently Wang et al. [22"] described a protein inhibitor of the primary sporulation kinase KinA that was encoded in an operon of genes of unknown function but regulated by nitrogen availability. This anti-kinase inhibitor, KipI, inhibited the autophosphorylation of KinA but not the phosphotransferase reaction. Another gene of the operon codes for an anti-anti-kinase protein, KipR, which neutralizes KipI activity. Thus, KinA inhibition by this system is regulated by the transcription of the kip gene operon and by the dynamics of the interaction between KipI and KipR. Surprisingly, it was found that KipI interacts with the catalytic domain of KinA, specifically the ATP-binding site, not the sensor domain. This is the first example of two-component signal transduction inhibitors that act on the catalytic domain of sensor kinases to counteract the signals activating the sensor domain of these kinases.
Overview of sporulation initiation in B. subtilis It is now clear that the phosphorelay signal transduction pathway provides many access points for regulation by inhibitors induced by cellular processes presumably antithetical to sporulation (Figure 1). This includes at least two phosphatases for Spo0F-P regulated by different inducers and a phosphatase for Spo0A-P. T h e cffectors of KinA and KinB are unknown but is now known that
172
Cell regulation
KinB must be sensing something in the periplasm [23[. Thus, we now have an appreciation of the mechanisms that regulate the level of Spo0A-P but the cellular functions and molecules that regulate the mechanisms remain obscure.
Sensor kinases in development Development into spores in Al),xococcus xanthus is a communal affair in which 105 cells form an organized mound or fruiting body inside which the spores are formed. Although ,.11. xanthus is Gram negative, it shares with B. subti/is and lower eukaryotes the induction of sporulation by nutrient limitation. In ,.11. xanthus the
sasS gene codes for a transmembrane sensor histidine kinase that regulates early developmental gcne expression [24]. It is thought that this kinase may phosphorvlate a NtrC-like regulator that activates transcription of sigma-54 promoters known to be expressed during early development [13]. A gene, fruA, has been identified that encodes a transcriptional regulator required for sporulation and normal fruiting body morphogenesis [25]. FruA appears to be structurally similar to response regulators of two-component systems and may be activated by phosphorylation. Typical two-component histidine kinases have been found to be required for preventing gene expression and sporulation in Dico,ostelium [26",27"] and
Figure 1
Cell membrane
Cytoplasm KipR
KinA ATP
ADP
<~
'
Kipl , ~
l-R [Inactive]
KinA-~) KapB KinB-(~
) Import
O F - ( ~ ~
OB-(~ %"-&'~"--'~ OA-(~
~
PhrA
RapA
L
L.~)
r
spollG
|l
I
PhrA
Current Opinion in Microbiology
Regulatory interactions in the phosphorelay controlling sporulation in B. subtilis. Phosphorylated OA is a transcription activator of many genes, including spollG. Its phosphorylation level and activity are subject to dephosphorylation by OE phosphatase and by RapA and RapB phosphatases that dephosphorylate OF-P. The RapA phosphatase is regulated by a peptide product of the phrA locus processed after export to the exterior of the cell. Phosphate input by KinA is regulated by an anti-kinase, Kipl, and an anti-anti-kinase, KipR. KinB is located in the cellular membrane and its activity is controlled by KapB. Circled P, phosphorylation.
Initiation of bacterial development Hoch
conidiophore development in Neurospora crassa [28,29"]. Thus, signal transduction by two-component systems is likely to play a major role in environmental sensing and development in a variety of bacteria and sporulating fungi.
173
the mechanisms are exceedingly complex and precisely coordinated and we have really only had a glimpse of this machinery.
Acknowledgements GTPases in development Era and Obg GTP-binding proteins with GTPase activity are enigmatic components of bacterial cells thought to have roles in signal transduction because that is the role of similar proteins in higher organisms [30]. No one has defined what these proteins do in bacteria. In B. subtilis, the Obg protein is essential for growth and the initiation of sporulation but not for later stages of sporulation [31]. An Ohg homolog has been identified in Streptomyces species and increasing the cellular content of Obg from a multicopy plasmid repressed aerial mycetium development [32]. What pathway or process is being affected by these proteins? A low molecular weight GTPase, MglA, similar to the Ras/Rab/Rho superfamily required for multicelhdar development and gliding mobility has been found in ,.11. xanthus [33"']. Surprisingl'> the yeast GTPase Sarlp is able to complement the sporulation defect of an mg/A deletion. An undefined second site mutation, rpm, allowed Sarlp to suppress both developmental and motility defects of the deletion strain. Because the role of Sarlp is also unknown, the functions of MglA in motility and development remain murky.
Butyrolactone-regulating factors in
$treptomyces development Streptomyces species produce a family of butyrolactone autoregulators that control differentiation and secondary metabolism. T h e regulators can be classified into three types on the basis of structural differences. Receptors for each class, A, IM-2 and VG, have now been cloned and characterized [34]. These receptors have homology to the TetR family of transcriptional repressors and are specific for each butyrolactone. T h e A-factor butyrolactone is essential for aerial mycetium formation and streptomycin production in Streptomyces gHseus [351. This phenotype of an A-factor mutant may be reversed by the introduction of a Ser/Thr kinase gene from Streptomt'ces coe/icoloz, suggesting that the kinase is part of the A-factor regulatory network [35].
Conclusions T h e only feature that all microbial development seems to have in common is that induction of the process occurs under conditions of nutrient deprivation or high cell density. Many of the organisms appear to place some induction control on molecules that may signal cell density such as A-factor or butyrolactones, what the organism had in mind for this type of regulation can only be guessed at. At this point, most microbial development is still at t h e stage o f d e f i n i n g m e c h a n i s m s o f control r a t h e r t h a n w h a t is c o n t r o l l i n g t h e m e c h a n i s m : h o w e v e r , it is clear t h a t
The author wnuld like to acknowledge the advice of Heidi Kaplan and Kcith Chatcr in idcntit'ying papers of interests.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ,, of outstanding interest 1.
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2.
Wu U, Lewis PJ, AIImansberger R, Hauser PM, Errington J: A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis. Genes Dev 1995, 9:1316-1326.
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Wu U, Errington J: Septal localization of the SpolllE chromosome partitioning protein in Bacillus subtilis. EMBO J 1997, 16:2161-2169.
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5. •
Sharpe ME, Errington J: The Bacillus subtilis soj-spoOJ locus is required for a centromere-like function involved in prespore chromosome partitioning. Mo/Microbio/1996, 21:501-509. This work presages the role of the spo0J proteins in mitosis. 6. o•
Webb CD, Telernan A, Gordon S, Straight A, Belmont A, Lin DC, Grossman AD, Wright A, Losick R: Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subti/is. Cell 1997, 88:667-674. This article describes a very clever method for labelling the origin of chromosome replication with /ac operators and it demonstrates the behavior of origins in mitosis. 7. •,
Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Ceil 1997, 88:675-684. An excellent demonstration of the functions of ParA and ParB in chromosome segregation and mitosis. ParB was found to bind to sequences adjacent to the origin of replication and this region was tethered to the poles of the cell during a specific time of the cell cycle. ParA also segregated to the poles suggesting it was part of the mitotic appartus. 8. *•
Glaser P, Sharpe ME, Raether B, Perego M, Ohlsen KL, Errington J: Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev 1997, 11:1160-1168. The Spo0J protein of Bacillus subtih's is equivalent to the ParB of Caulobacter crescentus and was shown to localize close 1o the cell poles using immunofluorescence and a GTP fusion to Spo0J. These loci replicate in concert with the initiation of new rounds of DNA replication and they function to position the oriC region of the chromosome providing evidence for a mitotic-like apparatus. 9. •
Lin DCH, Levin PA, Grossman AD: Bipolar localization of a chromosome partition protein in Bacillus subtilis. Proc Nat/ Acad Sci USA 1997, 94:4721-4726. An independent study showing the segregation of Spo0J proteins. See annotation [8*']. 10.
Lewis PJ, Errington J: Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and colocalization with the Spo0J partitioning protein. Mo/Microbiol 1997, 25:945-954.
11.
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12.
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13.
Keseler IM, Kaiser D: Sigma 54, a vital protein for Myxococcus xanthus. Proc Natl Acad Sci USA 1997, 94:1979-1984.
1 ?4
Cell regulation
14. 6.
Domian IJ, Quon KC, Shapiro L: Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the Gl-to-S transition in a bacterial cell cycle. Ceil 1997, 90:415-424. This article demonstrates the regulatory modifications of an essential transcription regulator for the cell-cycle and suggests that cell cycle regulation in bacteria may be similar to that in eukaryotes. This may be what everyone is researching in 1998. 15. Wheeler RT, Shapiro L: Bacterial chromosome segregation: is e° there a mitotic apparatus? Cell 1997, 88:577-579. An insightful review of mitosis in bacteria and of references [6"*,7"']. 16.
Burbulys D, Trach KA, Hoch JA: The initiation of sporulation in Bacillus subtilis is controlled by a multicomponent phosphorelay. Ceil 1991, 64:545-552.
17.
Ohlsen KL, Grimsley JK, Hoch JA: Deactivation of the sporulation transcription factor Spo0A by the SpoOE protein phosphatase. Proc Nat/Acad Sci USA 1994, 91:1756-1760.
18.
Perego M, Hanstein CG, Welsh KM, Djavakhishvili T, Glaser P, Hoch JA: Multiple protein aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in Bacillus subtilis. Ceil 1994, 79:1047-1055.
19. ••
Perego M, Hoch JA: Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis. Proc Nat/Acad Sci USA 1996, 93:1549-1553. This reseach showed that a phosphatase regulating development was controlled by a peptide which is first secreted and subsequently reimported by the oligopeptide permease. These studies show that the peptide functioned by inhibition of phosphatase activity not by regulation of phosphatase gene expression. 20. ••
Perego M: A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay. Proc Nat/Acad Sci USA 1997, 94:8612-8617. A direct demonstration of the in vitro inhibition of protein aspartate phosphatases by regulatory peptides and an exploration of the specificity determinants. This work forms the basis for the hypothesis that peptides communicate the status of extramembranous events to the cell. Lazazzera BA, Solomon JM, Grossman AD: An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell 1997, 89:917-925. This paper demonstrates that producing the mature peptides that regulate phosphatases inside the cell obviates the need for secretion and importation. These studies confirm that the oligopeptide permease is responsible for import of the peptides.
24.
Yang C, Kaplan HB: Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression. J Bacterio/1997, 179:7759-7767
25.
Ogawa M, Fujitani S, Mao X, Inouye S, Komano T: FruA, a putative transcription factor essential for the development of Myxococcus xanthus. Mo/ Microbio/1996, 22:757-767
26. .
Wang N, Shaulsky G, Escalante R, Loomis WF: A twocomponent histidine kinase gene that functions in Dictyostelium developmenL EMBQ J 1996, 15:3890-3898. A two-component kinase has been identified in Dictyostefium development, proof that it is not just for B. subti/is development anymore. See also [27"]. 27. •
Schuster SS, Noegel AA, Oehme F, Gerisch G, Simon MI: The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium. EMBO J 1996, 15:3880-3889. See annotation [26°]. 28.
29. •
Alex LA, Borkovich KA, Simon Ml: Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase. Proc Nat/ Acad Sci USA 1996, 93:3416-3421. A two-component kinase in the hyphal development of fungi: where will it end? Agene coding for a histidine kinase with a response regulator domain was found to be involved in osmoregulation and hyphal development. Deletion of the gene for this kinase results in aberrant hyphal structure that is enhanced under conditions of osmostress. 30.
Pillutla RC, Ahnn J, Inouye M: Deletion of the putative effector region of Era, an essential GTP-binding protein in Escherichia coil, causes a dominant-negative phenotype. FEMS Microbiol Lett 1996, 143:47-55.
31.
Kok J, Trach KA, Hoch JA: Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg. J Bacteriol 1994, 176:7155-7160.
32.
Okamoto S, Itoh M, Ochi M: Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation. J Bacteriol 1997, 179:170-179.
21. •
22. •-
Wang L, Grau R, Perego M, Hoch JA: A novel histidine kinase inhibitor regulating development in Bacillus subtilis. Genes Dev 199"7, 11:2569-2579. A demonstration of a new type of two-component kinase inhibitor targeted to the catalytic domain and its regulation by an anti-anti-kinase. 23.
Dartois V, Liu J, Hoch JA: Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis. Mo/ Microbio/1997, 25:39-51.
Schumacher MM, Enderlin CS, Selitrennikoff CP: The osmotic1 locus of Neurospora crassa encodes a putative histidine kinase similar to osmosensors of bacteria and yeast. Curr Microbio/1997, 34:340-347.
33. •.
Hartzell PL: Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase. Microbiology 1997, 94:9881-9686. A fascinating demonstration of the complementation of a M. xanthus GTPase with a yeast counterpart. 34.
Waki M, Nihira T, Yamada Y: Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Strepfomyces sp. strain FRI-5. J Bacteriol 1997, 179:5131-5137.
35.
Ueda K, Umeyama T, Beppu T, Horineuchi S: The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2). Gene 1996, 169:91-95.