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A fundamental question in developmental biology is how morphogenesis is coordinated with cell cycle progression. In Caulobacter crescentus, each cell cycle produces morphologically distinct daughter cells, a stalked cell and a flagellated swarmer cell. Construction of both the flagellum and stalk requires the alternative sigma factor RpoN (σ54). Here we report that a σ54-dependent activator, TacA, is required for cell cycle regulated stalk biogenesis by collaborating with RpoN to activate gene expression. We have also identified the first histidine phosphotransferase in C. crescentus, ShpA, and show that it too is required for stalk biogenesis. Using a systematic biochemical technique called phosphotransfer profiling we have identified a multicomponent phosphorelay which leads from the hybrid histidine kinase ShkA to ShpA and finally to TacA. This pathway functions in vivo to phosphorylate and hence, activate TacA. Finally, whole genome microarrays were used to identify candidate members of the TacA regulon, and we show that at least one target gene, staR, regulates stalk length. This is the first example of a general method for identifying the connectivity of a phosphorelay and can be applied to any organism with two-component signal transduction systems.
In many organisms, programs of morphogenesis and development are temporally coupled to cell cycle progression, yet the molecular basis of this coupling remains poorly understood, particularly in bacteria. Caulobacter crescentus has emerged as a tractable model system for studying the regulation of these fundamental processes as cell cycle progression in this organism is accompanied by a series of morphological transitions culminating in the production of two distinct daughter cells, a swarmer cell and a stalked cell (Ohta et al., 2000; Ryan and Shapiro, 2003; Skerker and Laub, 2004). Motile swarmer cells possess a single polar flagellum and polar pili, and they cannot initiate DNA replication. Swarmer cells differentiate into stalked cells by shedding their polar flagellum, retracting the pili, and synthesizing a stalk at the former site of the flagellum. This motile-to-sessile transition accompanies a G1-S transition and the initiation of a single round of DNA replication. During the ensuing S-phase and a brief G2 phase, the predivisional cell continues to extend the polar stalk, while also building a new polar flagellum and pili opposite the stalked pole. An asymmetric cell division then ensues, producing a new swarmer and new stalked cell (see also Fig. 7).
The polar stalk of Caulobacter has been suggested to serve a number of important functions. The adhesive polysaccharide substance called holdfast at the distal tip of the stalk allows cells to bind to abiotic surfaces or to other cells (Poindexter, 1964; Kurtz and Smit, 1994; Smith et al., 2003). As Caulobacter is an aquatic, aerobic bacterium, the stalk has also been suggested to affect buoyant density and help cells stay associated with the air–water interface (Poindexter, 1978). Finally, the stalk may play a significant role in nutrient acquisition. Cells starved for phosphate extend their stalks up to 10 or 20 times the length of the cell body, possibly as a mechanism to increase surface area and nutrient uptake ability (Poindexter, 1964; 1984; Gonin et al., 2000).
Little is known about the regulation of stalk biogenesis. However, many other regulatory processes central to cell cycle progression and morphogenesis in Caulobacter are carried out by two-component signal transduction systems (Ohta et al., 2000; Skerker and Laub, 2004). These signalling systems, comprised of histidine kinases and response regulators, are widespread in the prokaryotic kingdom, as well as being found in plants, yeast and slime moulds (Hoch and Silhavy, 1995). The Caulobacter genome encodes 62 histidine kinases and 44 response regulators (Nierman et al., 2001; Skerker et al., 2005). The canonical view of these signalling systems involves a sensor histidine kinase which autophosphorylates on a conserved histidine residue and then transfers the phosphoryl group to an aspartate residue on the receiver domain of a response regulator (Fig. 1A). Phosphorylation of the receiver domain activates the output domain, which often has the ability to bind DNA, thereby coupling signalling pathways to changes in gene expression and the execution of various cellular events.
Complicating this simple paradigm for two-component signalling are hybrid histidine kinases in which a histidine kinase contains a response regulator receiver domain at its C-terminal end. These hybrid histidine kinases often participate in more complex pathways called phosphorelays (Fig. 1A). In such pathways, hybrid kinases first autophosphorylate on a conserved histidine residue in the kinase domain and then transfer the phosphoryl group intramolecularly to a conserved aspartate in its receiver domain. The phosphoryl group is then transferred to another conserved histidine on a protein called a histidine phosphotransferase, which can in turn transfer its phosphoryl group to an aspartate on a diffusible response regulator. Unlike histidine kinases and response regulators, histidine phosphotransferases show little sequence identity to one another, with conservation of only a few critical residues and a four-helix bundle structure (Kato et al., 1997; Song et al., 1999; Xu and West, 1999).
Previous genetic screens as well as a systematic deletion of each of the 106 two-component genes in Caulobacter have identified 39 genes which are required for normal cell cycle progression or morphogenesis (Skerker et al., 2005). Of these, 19 were impaired in motility and nine had defects in stalk biogenesis. Many of those involved in motility had been previously identified as regulators of flagellar biogenesis, the best understood aspect of morphogenesis in Caulobacter. The assembly of the polar flagellum in predivisional cells is controlled by a four-tiered transcriptional cascade (Ramakrishnan et al., 1994; Gober and England, 2000). This cascade is set in motion by the DNA-binding response regulator CtrA (Quon et al., 1996). The phosphorylation and activity of CtrA are under extensive cell cycle control, coupling flagellar biogenesis to cell cycle progression (Quon et al., 1996; Domian et al., 1997). CtrA directly regulates the expression of at least 95 genes, including rpoN, which encodes the σ54 subunit of RNA polymerase (Laub et al., 2002). rpoN mutants are non-motile and stalkless, suggesting that σ54 regulates genes involved in flagellum and stalk biogenesis (Brun and Shapiro, 1992).
σ54-dependent gene expression typically requires a phosphorylated transcriptional activator of the NtrC-family of response regulators (Kustu et al., 1989). In Caulobacter, flbD encodes a σ54-dependent activator which is induced at approximately the same time as rpoN, and together with RpoN, activates expression of genes required late in flagellar assembly (Ramakrishnan et al., 1991). FlbD also represses genes required early in flagellar assembly, and thus coordinates the timing of multiple steps in flagellum assembly (Wingrove et al., 1993; Benson et al., 1994; Wingrove and Gober, 1994). However, unlike rpoN, flbD mutants are not impaired in stalk synthesis, suggesting that another NtrC-like response regulator functions with rpoN to regulate stalk biogenesis.
In a systematic deletion of two-component signalling genes the NtrC-like regulator tacA was found to lack stalks (Skerker et al., 2005), suggesting it regulates stalk biogenesis. In that study and previous genetic screens, deleting the histidine kinase pleC was also found to produce stalkless cells (Sommer and Newton, 1989; Skerker et al., 2005). PleC, however, exclusively phosphorylates the response regulators DivK and PleD, and not TacA (Skerker et al., 2005). In the systematic deletion study no other deletions of canonical histidine kinases produced a stalkless phenotype. However, the hybrid kinase CC0138 was found to be stalkless, suggesting that a phosphorelay controls phosphorylation of the σ54-activator TacA. Here, using genetics and a systematic biochemical method called phosphotransfer profiling, we describe the identification of ShpA, the first histidine phosphotransferase in Caulobacter, and demonstrate that it is central to a phosphorelay which regulates stalk biogenesis by controlling the activity of TacA. Using whole-genome DNA microarrays, we identify the likely target genes of TacA. Our results provide evidence for a transcriptional cascade that regulates stalk biogenesis and suggest a mechanism for the cell cycle timing of stalk synthesis.
Identification of C. crescentus histidine phosphotransferases
A systematic study of all C. crescentus two-component signalling genes identified two genes whose deletion produced stalkless cells, the gene CC0138 which encodes a hybrid histidine kinase and CC3315 which encodes a σ54-dependent response regulator named TacA (Skerker et al., 2005). The discovery of these mutants suggested that a phosphorelay may control stalk formation in C. crescentus. However, phosphorelays require histidine phosphotransferases to shuttle phosphoryl groups from hybrid kinases to response regulators (Fig. 1A), and none were identified in the initial annotation of the C. crescentus genome based on sequence homology methods (Nierman et al., 2001). To identify putative phosphotransferases in the C. crescentus genome, we searched for genes predicted to encode proteins bearing other common features. Specifically, we sought proteins with (i) fewer than 250 amino acids, (ii) greater than 70% predicted alpha helical secondary structure, and (iii) the conserved motif HXXKG within a predicted alpha helix. Two genes satisfied these criteria, CC1114 and CC1220. This report focuses on the gene CC1114 which is predicted to encode a protein of 112 amino acids with high alpha helical content (Fig. 1B). The primary sequence of CC1114 was successfully threaded onto the known four-helix bundle structures of three monomeric histidine phosphotransferases: Ypd1 from Saccharomyces cerevisiae (Xu and West, 1999), LuxU from Vibrio harveyi (Ulrich et al., 2005), and the phosphotransferase domain of ArcB from Escherichia coli (Kato et al., 1997) (Fig. 1C). These threaded structural alignments further predicted that His-56 of CC1114 lies within the middle of an alpha helix and is solvent exposed. Homologues of CC1114 are found in the genomes of closely related alpha-proteobacteria (Fig. S1). In sum, these bioinformatic analyses suggested that CC1114 may encode a histidine phosphotransferase.
CC1114 has histidine phosphotransferase activity in vitro
To determine whether the CC1114 protein exhibits phosphotransferase activity, we tagged CC1114 with a His6-tag and purified the protein by affinity chromatography to greater than 90% homogeneity. To determine whether CC1114 can receive a phosphoryl group from a hybrid histidine kinase, we incubated CC1114 with the purified kinase and receiver domains from each of four hybrid kinases which possess high in vitro activity: CC0138, CckA, CC3102 and CC3219. We used separate kinase and receiver domains for each hybrid kinase because full-length constructs had little to no activity (data not shown). Reactions were initiated by the addition of [γ-32P]-ATP, incubated for 30 min, and then resolved by SDS-PAGE (Fig. 2A). In each reaction, radiolabel was incorporated into the kinase domain by autophosphorylation and subsequently transferred to the corresponding receiver domain. For the reactions with CC0138 and CC3102, significant accumulation of label was also found at a position corresponding to CC1114. Incubation of CC1114 with the CC0138 kinase domain in the absence of the CC0138 receiver domain was indistinguishable from incubating the CC0138 kinase domain alone (Fig. 2B, lane 1 vs. 4) indicating that (i) the receiver domain is required for phosphotransfer from the kinase domain to CC1114, and (ii) CC1114 does not have autophosphorylation activity. Together these observations support the conclusion that CC1114 is a histidine phosphotransferase.
CC1114 participates in a phosphorelay culminating in the phosphorylation of TacA
We previously developed an assay called phosphotransfer profiling which allows the rapid, systematic identification of response regulator targets of a given histidine kinase (Skerker et al., 2005). In this assay, one histidine kinase is systematically tested in vitro for its ability to transfer a phosphoryl group to each purified response regulator encoded in the genome. As histidine kinases exhibit a large kinetic preference in vitro for their in vivo cognate substrates, kinase-regulator pairs are easily identified by testing phosphotransfer at multiple time points and identifying the kinetically preferred substrate(s) (Skerker et al., 2005). Here, we adapted this assay (also see Fig. S2) to examine the substrate specificity, and hence probable in vivo partners, of CC1114. CC1114 was first phosphorylated (producing CC1114∼P) and then split and incubated individually for 2 min with each of the 44 purified, full-length response regulators encoded in the C. crescentus genome (Skerker et al., 2005). Phosphorimaging demonstrated a single preferred target of CC1114∼P, the response regulator TacA (Fig. 2C).
Next, we wished to identify the hybrid kinase which phosphorylates CC1114. In vivo hybrid histidine kinases can pass a phosphoryl group to a phosphotransferase or vice versa, with the directionality typically driven by mass-action (Uhl and Miller, 1996; Georgellis et al., 1998). We took advantage of this reversibility to identify the hybrid kinases which interact with CC1114, by profiling phosphotransfer from CC1114∼P to the purified receiver domains of each of the 27 hybrid kinases encoded in C. crescentus (Fig. S3). With phosphotransfer reaction times of 2 min, two receiver domains, from the hybrid kinases CC0138 and CC0921, were efficiently phosphorylated, resulting in depletion of radiolabel from CC1114∼P (Fig. 2D). However, with a shorter reaction time (Fig. 2E), CC1114 exhibited a kinetic preference for phosphotransfer to the CC0138 receiver domain. Furthermore, the rate of transfer to the CC0138 and TacA receiver domains was approximately equal, suggesting that these are the kinetically preferred and hence most likely in vivo partners for CC1114 (Fig. 2E). Finally, as a control, we examined the phosphotransfer profile of the CC0138 kinase domain with respect to each of the 44 soluble, full-length C. crescentus response regulators. This experiment verified that CC0138 does not directly phosphorylate TacA or any response regulator other than its own receiver domain (data not shown).
Taken together, the phosphotransfer profiles suggest that CC1114 mediates a phosphorelay between the hybrid kinase CC0138 and the response regulator TacA (Fig. 2F). To verify the complete biochemical pathway, we mixed all components with [γ-32P]-ATP and incubated reactions at 30°C (Fig. 2G). Phosphorimaging showed that label accumulated mostly in TacA (Fig. 2G, lane 4). Excluding the CC0138 receiver domain (Fig. 2G, lane 2) or CC1114 (Fig. 2G, lane 3) from the reaction eliminated phosphorylation of TacA. These data support the conclusion that autophosphorylation of CC0138 leads to a phosphorelay through its own receiver domain and CC1114, culminating in the phosphorylation of TacA.
CC0138, CC1114 and TacA control stalk biogenesis and cell division
The phosphotransfer experiments suggested that CC1114 mediates an exclusive phosphorelay which leads to phosphorylation of TacA. As deletion strains for both tacA and CC0138 were found previously to lack stalks (Skerker et al., 2005), we predicted that a CC1114 deletion should also be stalkless. We therefore constructed a strain in which almost the entire coding region of CC1114 was replaced with a tetracycline resistance cassette. Examination of this strain by light microscopy confirmed that ΔCC1114, like ΔCC0138 and ΔtacA, is stalkless in rich (Fig. 3A, D, G and J) and minimal media (data not shown). However, deletion strains for each phosphorelay component were able to synthesize stalks when grown in low phosphate medium, a condition known to trigger extensive stalk growth (Poindexter, 1964; 1984; Gonin et al., 2000), suggesting that these strains are impaired in the regulation of stalk biogenesis, not stalk synthesis per se (Fig. 3C, F, I and L). Each deletion strain also appeared mildly filamentous with at least 20% of cells showing obvious cellular elongation relative to wild type, suggesting a defect in cell division or control of cell growth. Motile cells of ΔCC0138, ΔCC1114 and ΔtacA were easily observed by microscopy, but due to elongation cells did not swim as rapidly as wild type. Consistent with this observation, each phosphorelay mutant showed partial defects in a swarm plate motility assay (Fig. 3M). ΔCC0138, ΔCC1114 and ΔtacA each exhibited a swarm size intermediate between that of the wild-type CB15N and the non-motile strains ΔflbD and rpoN::Tn5, presumably due to the inability of the filamentous, mutant cells to swim efficiently through the agar.
The mutant phenotypes of ΔCC0138, ΔCC1114 and ΔtacA were each complemented by providing the deleted gene on a plasmid (Fig. 4A, C and H), demonstrating that the cell division and stalk phenotypes were due to a single gene deficiency in each case. ΔCC1114 cannot however, be complemented by an allele of CC1114 bearing the H56A mutation (Fig. 4B), consistent with the prediction that His-56 is the sole phosphorylation site on CC1114. Moreover, purified CC1114(H56A) also did not exhibit phosphotransferase activity in vitro (data not shown).
In sum, deleting any component of the CC0138-CC1114-TacA phosphorelay led to a minor defect in cell division or control of cell growth resulting in elongated cells, and a major defect in stalk biogenesis. As growth, division, and stalk biogenesis each involve the coordination of peptidoglycan and cell membrane metabolism (Wagner et al., 2005), a single deficiency may underlie all of the morphological phenotypes of ΔCC0138, ΔCC1114 and ΔtacA. Moreover, the similarity of mutant phenotypes for CC0138, CC1114 and tacA supports the notion that they act in the same signalling pathway in vivo, as suggested by the in vitro results demonstrating a phosphorelay comprised of these proteins. As the major defect of the deletion strains is in stalk biogenesis we have named CC0138 shkA for stalk biogenesis histidine kinase A and named CC1114 shpA for stalk biogenesis histidine phosphotransferase A.
The constitutively active allele tacA(D54E) rescues ΔshkA, ΔshpA and ΔtacA
The data presented thus far suggest that ShkA and ShpA function in vivo to phosphorylate TacA. We therefore predicted that expression of a constitutively active form of tacA, tacA(D54E), should rescue deletion of shkA or shpA. In the NtrC family of response regulators, which includes TacA, changing the active site aspartate to a glutamate often mimics phosphorylation of the response regulator and hence renders its activation independent of upstream kinases or phosphorelays (Klose et al., 1993). We therefore placed a tacA(D54E) allele on the vector pJS71 under control of the xylose-inducible promoter PxylX (Meisenzahl et al., 1997) and transformed this plasmid into the strains ΔshkA, ΔshpA and ΔtacA. Each strain was grown in rich medium supplemented with glucose. As the pJS71 vector is a relatively high copy vector, growth in the presence of the repressor glucose leads to a low level of expression from PxylX but prevents overexpression. For ΔshkA, ΔshpA and ΔtacA, expression of tacA(D54E) rescued both the cellular elongation and stalk defects of the mutant strains (Fig. 4E, G and I). In contrast, expression of wild-type tacA from the same plasmid rescued ΔtacA (Fig. 4H), but did not rescue the mutant phenotypes of ΔshkA or ΔshpA (Fig. 4D and F). Even overexpression of wild-type tacA, by growth in 0.1% xylose, did not rescue the ΔshkA and ΔshpA mutants (data not shown). These data support the conclusion that the primary in vivo function of ShkA and ShpA is to phosphorylate, and hence activate, TacA.
Next, we examined the effect of overproducing TacA(D54E). Wild-type cells harbouring the PxylX-tacA(D54E) allele on pJS71 were grown in the presence of either glucose or xylose. In glucose, these cells appeared wild type in terms of morphology (Fig. 5A) and growth rate (data not shown). However, in xylose the cells grew almost twice as slowly as in glucose and rapidly became filamentous. Cells had abnormal stalks that were shorter or longer than wild type and in some cases appeared only as a small bud or protrusion from the cell pole (Fig. 5B). These growth and stalk defects could be the result of high levels of constitutively active TacA and/or the synthesis of active TacA at the wrong time during cell cycle progression. Together, the overexpression and deletion analyses indicate that TacA activity must be tightly regulated during the Caulobacter cell cycle to ensure proper stalk biogenesis.
Identifying the TacA regulon with DNA microarrays
TacA is a member of the NtrC family of response regulators, also known as enhancer-binding proteins (EBPs), which typically function to activate σ54-dependent gene expression. σ54 is encoded in Caulobacter by the gene rpoN and is required for both flagellar and stalk biogenesis (Brun and Shapiro, 1992). FlbD was identified as the EBP response regulator which works with σ54 in predivisional cells to regulate the expression of flagellar genes (Ramakrishnan et al., 1991; Wingrove et al., 1993; Benson et al., 1994; Wingrove and Gober, 1994). Based on our results, we hypothesize that TacA collaborates with σ54 to control genes involved in regulating stalk biogenesis. To identify possible target genes, we used whole genome DNA microarrays to interrogate the expression patterns of TacA phosphorelay mutants (ΔshkA, ΔshpA, ΔtacA) and a rpoN disruption mutant rpoN::Tn5 (Fig. 6). To partition the σ54 regulon into FlbD- and TacA-dependent genes, we also examined a ΔflbD strain (Fig. 6).
Strains were grown to mid-log phase (OD600 ∼0.4) in rich medium, then RNA was harvested and used to probe whole-genome DNA microarrays. Each mutant strain was compared with the wild-type CB15N and each comparison done in duplicate or triplicate with results averaged. Genes whose expression level changed at least twofold in rpoN::Tn5 and in any one of the phosphorelay or flbD mutants were selected for further analysis (Fig. 6). Visual inspection of expression profiles for each mutant relative to wild type, clearly demonstrated the similarity of ΔshkA, ΔshpA, ΔtacA and rpoN::Tn5 (Fig. 6). Correlation coefficients for each pairwise comparison among these mutants were greater than 0.73. Similarly high correlation coefficients have been seen for mutants of other two-component signalling genes which function in the same pathway (Jacobs et al., 2003). These results thus add further support to the biochemical and genetic data demonstrating that ShkA-ShpA-TacA constitutes a phosphorelay which controls gene expression through phosphorylation of TacA.
We predicted that the TacA target genes involved in stalk biogenesis would be similarly affected in ΔshkA, ΔshpA, ΔtacA and rpoN::Tn5, each of which lacks stalks, but unchanged in the ΔflbD mutant which is non-motile, but retains stalks. In total, 30 genes satisfied these criteria, two of which are consistently downregulated in each stalkless mutant and 28 upregulated in each. The two downregulated genes contained σ54 binding sites (TGGCCC-N5-TTGC) (Wu et al., 1995) in their upstream regulatory regions, suggesting that each is a direct target of TacA and σ54. CC3218 has no predicted function based on sequence homology but is transcriptionally cell cycle-regulated (Laub et al., 2000) with expression highest in stalked cells and again in late predivisional cells. The other putative TacA target, CC2251, is predicted to encode a transcription factor of the Cro/CI family. During wild-type cell cycle progression CC2251 mRNA levels peak in predivisional cells, shortly after tacA expression peaks, suggesting that these regulators may form a transcriptional cascade which controls stalk biogenesis (Fig. 7).
Although EBP response regulators such as TacA typically activate gene expression, some can directly repress genes, independent of σ54 (Wingrove et al., 1993; Benson et al., 1994; Wingrove and Gober, 1994). Hence to identify genes which may be directly repressed by TacA, we searched for genes whose expression was significantly upregulated in ΔtacA, but unaffected in the rpoN mutant (Table S1). The only gene fitting these criteria was CC3314, which is annotated as a ‘conserved hypothetical’ and reads divergently from tacA itself. All other genes are similarly upregulated in rpoN::Tn5 and each of the phosphorelay mutants, suggesting that they are regulated indirectly by TacA, perhaps through the transcription factor CC2251. The vast majority of these genes have no annotated function, but their roles, if any, in stalk biogenesis can now be explored.
staR (CC2251) expression is regulated by the TacA phosphorelay and regulates stalk biogenesis
Microarray analysis identified at least two genes, CC2251 and CC3218, as probable direct targets of TacA. To determine their roles, if any, in stalk biogenesis, we constructed deletion and overexpression strains for each gene. Deletion and overexpression of CC3218 had no obvious phenotype (data not shown). Deletion of CC2251 did not eliminate stalks, but the average stalk length for ΔCC2251 cells (0.68 µm ± 0.24, Fig. 5F) was significantly shorter than wild type (1.26 µm ± 0.32, Fig. 5E). Conversely, over-expression of CC2251 from a xylose-inducible promoter on the low-copy vector pMR20 resulted in cells with stalks that were highly variable and longer on average (1.95 µm ± 0.89, Fig. 5G) than wild type. Overexpression of CC2251 from a high-copy (pJS71) vector also produced longer stalks in addition to a growth defect and filamentous cells (Fig. 5D). This growth defect and filamentation was similar to the phenotype of TacA(D54E) overexpression (Fig. 5B), consistent with CC2251 being a direct target of TacA. Based on these observations we have named CC2251 staR for stalk biogenesis regulator. We note though, that because ΔCC2251 does not precisely phenocopy ΔtacA, there must be other TacA target genes which also help to control stalk biogenesis. Candidates from the DNA microarray data will need to be examined to fully account for the stalkless phenotype of tacA, shkA and shpA mutants.
Stalk biogenesis in Caulobacter
For Caulobacter, a developmental program is tightly coupled to cell cycle progression. This development includes three major morphogenetic events, biogenesis of a flagellum, polar pili and a stalk. While the regulation of flagellum assembly and, to a lesser extent, pili biogenesis have been worked out in mechanistic detail (Gober and England, 2000; Skerker and Shapiro, 2000; Viollier et al., 2002a,b), the regulation of stalk biogenesis has remained poorly understood. Here, we have identified a key signal transduction pathway responsible for stalk formation during the cell cycle and have begun outlining the transcriptional pathways involved. This genetic circuitry is summarized in Fig. 7A.
At the heart of this model is σ54 which is necessary for both flagellum and stalk biogenesis. As in other bacteria, σ54-dependent gene expression in Caulobacter requires an activator of the NtrC-family of response regulators, also known as enhancer-binding-proteins, or EBPs. Typically, phosphorylation of an EBP enables it to stimulate the closed-to-open complex transition of a promoter bound by RNA polymerase containing a σ54 subunit, thereby coupling activation of the signalling pathway to changes in gene expression (Kustu et al., 1989). The EBP response regulator FlbD is induced at the same time as RpoN, and together they regulate genes involved in flagellar biogenesis (Fig. 7B) (Wingrove et al., 1993; Benson et al., 1994; Wingrove and Gober, 1994). We have now identified TacA as the EBP which collaborates with RpoN to regulate genes involved in stalk biogenesis. Furthermore, we identified a phosphorelay which regulates stalk biogenesis by modulating the phosphorylation state of TacA: ShkA, a hybrid histidine kinase, first autophosphorylates and then initiates a phosphorelay in which a phosphoryl group is passed to its own receiver domain, then to the histidine phosphotransferase ShpA, and finally to the transcriptional regulator TacA (Fig. 7).
DNA microarray analysis identified two genes controlled by TacA which may help regulate stalk biogenesis: CC2251 (staR) and CC3218. The mRNAs for both staR and CC3218 are cell cycle-regulated with induction in late predivisional cells, immediately after the induction of tacA (M. T. Laub, unpubl. data) (Fig. 7B). StaR appears to play a role in controlling stalk length, as the deletion strain has stalks shorter than wild type, and overexpression leads to longer stalks (Fig. 5). However, as staR mutants do not completely lack stalks or exhibit cellular elongation like tacA mutants, other TacA targets remain to be identified and characterized. The microarray data presented here can now be explored to find these additional targets.
The coupling of stalk biogenesis to cell cycle progression is accomplished in large part by CtrA, the master cell cycle regulator. CtrA is regulated at multiple levels during cell cycle progression such that activity drops during the G1-S transition and then rapidly returns to peak levels as S phase proceeds (Domian et al., 1997). CtrA triggers both flagellum and stalk biogenesis by directly activating rpoN (σ54) and tacA (Laub et al., 2002). Taken together, we propose that stalk biogenesis, like flagellum biogenesis, is regulated by a transcriptional cascade: CtrA is induced after DNA replication initiation and directly activates synthesis of σ54 and TacA, which together activate the expression of stalk regulatory genes, including the transcription factor staR, which in turn probably controls the expression of yet other genes required for stalk biogenesis.
Precisely how PleC, another histidine kinase required for stalk biogenesis, feeds into the stalk regulatory circuit remains unknown. However, suppressor analyses place CtrA downstream of PleC, suggesting that the effect may be through regulation of CtrA activity (Ohta et al., 1992; Wu et al., 1998).
Stalk biogenesis is a cell cycle-regulated event and initially occurs in only one of the two daughter cells produced by cell division, raising the possibility that TacA activity is controlled spatially as well as temporally. FlbD, for example, is phosphorylated and active only in the nascent swarmer cell, where it activates expression of late flagellar genes and represses early flagellar genes (Gober and England, 2000). In the nascent stalked cell, FlbD is present but inactive; this pool of FlbD will be activated as the stalked cell develops, initiating early flagellar gene expression in the predivisional cell. Whether TacA activity is also asymmetrically regulated in a manner that restricts stalk biogenesis to a single daughter cell, the stalked cell, remains to be explored.
What signals lead to activation of TacA?
Why does Caulobacter use a phosphorelay to control stalk biogenesis? It has been suggested that phosphorelays allow the integration of multiple signals by providing numerous points of control. In Bacillus subtilis, a number of signals impinge on the phosphorelay controlling sporulation initiation by affecting the phosphorylation of different pathway components (Perego et al., 1994). Stalk biogenesis is a major developmental event in the life cycle of Caulobacter, and hence may also integrate a wide range of signals. However, the nature of the signal(s) controlling the ShkA-ShpA-TacA phosphorelay is not yet known. ShkA is predicted to be a soluble, cytoplasmic kinase, suggesting that the phosphorelay may respond to an internal cell cycle cue. Caulobacter thus appears to have at least two independent pathways for regulating stalk biogenesis, one dependent on an external cue, phosphate starvation, and one linked to cell cycle progression.
Identification of a histidine phosphotransferase
ShkA-ShpA-TacA is the first phosphorelay identified in C. crescentus as ShpA is the first histidine phosphotransferase identified in this organism. The C. crescentus genome likely encodes other histidine phosphotransferases. CC1220, identified here in the same computational screen as ShpA, is one candidate, but others almost certainly exist, particularly given that the Caulobacter genome encodes 27 hybrid histidine kinases. Hybrid kinases often function in phosphorelays like the one described here; however, in some cases the C-terminal receiver domain of a hybrid kinase may function as an auto-inhibitory domain. Intramolecular phosphorylation may relieve this inhibition, allowing the kinase to phosphorylate another, diffusible response regulator, as with the Agrobacterium tumefaciens hybrid kinase VirA (Chang et al., 1996).
Rapid, systematic mapping of complex phosphorylation cascades
To identify the probable in vivo cognate substrates for ShpA, we adapted a technique called phosphotransfer profiling (Skerker et al., 2005). By examining phosphotransfer from a kinase to each response regulator in parallel, and at multiple time points, kinetic preference is revealed and identifies the probable in vivo targets of the kinase (Skerker et al., 2005). Here, we hypothesized that histidine phosphotransferases would similarly exhibit a kinetic preference with respect to both soluble response regulators and the receiver domains of hybrid histidine kinases. A previous smaller scale study of two phosphorelays also suggested that histidine phosphotransferases exhibit kinetic preference in vitro (Perraud et al., 1998). Our phosphotransfer profiling experiments indeed demonstrated a kinetic preference for ShpA to transfer phosphoryl groups to or from the receiver domains of ShkA and TacA. Our genetic analyses (Figs 3 and 4) and DNA microarray results (Fig. 6) confirmed the in vivo relevance of these interactions. We suggest that histidine phosphotransferases in general may exhibit a global kinetic preference for interaction with their cognate substrates, similar to what has been observed for histidine kinases.
The global kinetic preference of histidine kinases for their cognate response regulator substrates means that the specificity of two-component signal transduction pathways is determined largely at a biochemical level; other factors probably function primarily to enhance this inherent specificity. Our results with ShpA suggest that these observations extend to histidine phosphotransferases and are general properties of two-component signalling pathways. We thus anticipate that our phosphotransfer profiling technique will be generally applicable to the mapping of phosphorelays, both in Caulobacter as well as in other organisms. Two-component signalling systems are prevalent throughout the bacterial kingdom as well as being present in fungi and plants (Stock et al., 2000), and the mapping of pathway connectivity among large numbers of signalling components remains a major challenge.
Many organisms couple developmental programs and morphogenesis to cell cycle progression. In the budding yeast S. cerevisiae, mating requires a morphological change called shmooing which prepares haploid cells for fusion. This morphological event is tied to cell cycle progression and can only occur during G1 phase. For B. subtilis the initiation of sporulation, a complex developmental program, is also tightly coupled to cell cycle events (Burkholder et al., 2001). The morphological events of flagellar, pili and stalk biogenesis in Caulobacter are similarly dependent on cell cycle progression. The identification of a complete signalling pathway controlling stalk biogenesis now opens the way to a more complete understanding of how this key morphological event is regulated in space and time during the Caulobacter cell cycle.
Bacterial strains, plasmids and growth conditions
Caulobacter crescentus strains were grown in peptone-yeast extract (PYE, rich medium) or M2G (minimal medium) at 30°C (Ely, 1991), supplemented with 3% sucrose, tetracycline (1 µg ml−1), kanamycin (25 µg ml−1), spectinomycin (25 µg ml−1), 0.1% glucose, or 0.1% xylose, as required. Low-phosphate growth experiments were performed as previously described (Quon et al., 1996). E. coli strains were grown at 30°C or 37°C in Luria–Bertani broth supplemented with carbenicillin (100 µg ml−1), chloramphenicol (30 µg ml−1), tetracycline (10 µg ml−1), kanamycin (50 µg ml−1) or spectinomycin (50 µg ml−1) as necessary. PYE swarm plates contained 0.3% bacto agar. Plasmids were transformed into C. crescentus by electroporation. Tetracycline marked deletion strains for CC1114, CC2251 and CC3218 were generated by a two-step recombination protocol (Skerker et al., 2005). pENTR clones of the 27 hybrid kinase receiver domains were constructed as previously described (Skerker et al., 2005). For in vivo expression of Caulobacter genes, pENTR plasmids for CC0138, CC1114, CC1114 (H56A), TacA, TacA(D54E), CC2251 and CC3218 were recombined with either a high-copy destination vector pHXM-DEST or a low-copy destination vector pLXM-DEST (Table S2), as necessary, to generate xylose-inducible constructs (Table S2). For overexpression experiments in Caulobacter, using pHXM-based vectors, saturated overnight cultures grown in PYE plus glucose were washed twice in PYE medium and then diluted to OD600 = 0.03 in either PYE plus glucose or xylose. For experiments using the low copy vector pLXM-CC2251 samples were grown overnight in PYE plus xylose prior to analysis.
Strains and plasmids generated are listed in Table S2. Primers and plasmids used to generate deletion strains, pENTR clones and point mutations are listed in Table S3. Site-directed mutagenesis was performed using the QuikChange protocol (Stratagene), with pENTR clones as templates. Morphology of mid-log phase cultures was imaged as described previously using DIC microscopy (Skerker et al., 2005). Stalks were visualized by staining with 5 µM Syto-9 (Molecular Probes).
Sequence analysis and homology modelling
Alpha-helical content was estimated using the Garnier algorithm within EMBOSS (http://emboss.sourceforge.net). A structure-based multiple sequence alignment of CC1114 against other known phosphotransferases was constructed using 3D-Coffee (Poirot et al., 2004). This alignment was used as input to Swiss-Model (Schwede et al., 2003) to predict the structure of CC1114 using the following templates: ArcB (PDB: 1FR0), Ypd1 (PDB: 1QSP) and LuxU (PDB: 1Y6D). ArcB provided the best alignment and the predicted CC1114 structure was analysed and displayed using UCSF Chimera (Pettersen et al., 2004).
Gateway cloning and protein purification
Escherichia coli thioredoxin (TRX)-His6 expression plasmids for all 44 response regulators and 27 hybrid kinase receiver domains were generated using recombinational cloning and purified as described previously (Skerker et al., 2005). CC1114 and CC1114(H56A) were expressed and purified as N-terminal His6-tagged proteins using the destination vector, pHIS-DEST. To construct pHIS-DEST, pET15b (Novagen) was digested with NdeI, blunted with T4 DNA polymerase, and the RfC Gateway vector conversion cassette (Invitrogen) was inserted. A His6-version of the TacA receiver domain (TacA-RD) was also constructed and purified, using pHIS-DEST. TacA-RD yielded a cleaner purification, and acted identical to full-length TacA in our assays, so we substituted this protein as necessary. Entry clones of the kinase domains of CckA, CC0138, CC3102 were recombined into pTRX-HIS-DEST or pHIS-MBP-DEST (for CC3219), and purified as either TRX-His6 or His6-MBP fusions. All purified proteins were stored in HKEDG buffer (10 mM HEPES-KOH, pH 8.0, 50 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT) and concentrations were normalized by densitometry.
Phosphotransfer profiling and phosphorelay biochemistry
We used a modified phosphotransfer profiling method (Skerker et al., 2005) to identify the cognate substrates for CC1114 (Fig. 2C and D, Fig. S2). All reactions and dilutions were performed in HKEDG buffer plus 5 mM MgCl2. First, we generated CC1114∼P by incubating 10 µM His6-CC1114 with 1 µM TRX-His6-CC0138-HK and 1 µM TRX-His6-CC0138-RD plus 1 µCi µl−1[γ-32P]ATP (∼6000 Ci mmol−1, Amersham Biosciences) for 30 min at 30°C. This reaction was then diluted 10-fold, and 5 µl of the mixture was added to 5 µl of buffer alone (as a control) or 5 µl of each purified response regulator or hybrid receiver domain (diluted to 5 µM) with incubations for 2 min at room temperature. The final concentrations were 0.5 µM for CC1114, and 2.5 µM for the phosphotransfer substrates. To demonstrate phosphorylation of CC1114 by various hybrid kinases (Fig. 2A), the following four autophosphorylation reactions (30 µl total volume) were performed: (i) TRX-His6-CckA (6 µM), (ii) TRX-His6-CC0138-HK (0.5 µM), (iii) TRX-His6-CC3102-HK (4.5 µM), or (iv) His6-MBP-CC3219-HK (20 µM) were incubated for 30 min at 30°C after addition of 30 µCi [γ-32P]ATP. Then, 1 µl of reaction A, 1 µl of reaction B, 4 µl of reaction C, and 9 µl of reaction D were mixed with 1 µM of the corresponding receiver domains of each histidine kinase, and, as necessary, with 10 µM of His6-CC1114 for 2 or 5 min, in a total volume of 10 µl. For analysis of CC1114 activity (Fig. 2B), reactions were performed the same as for phosphotransfer profiling except that various components were omitted before incubating 30 min at 30°C. For analysis of the complete TacA phosphorelay and kinetics (Fig. 2E and G), CC1114∼P was prepared as above, diluted, and then mixed with 5 µl of 5 µM His6-TacA-RD or other hybrid kinase receiver domains. All phosphotransfer reactions were stopped with 3.5 µl of 4 × SDS-PAGE sample buffer and analysed using 12% or 15% Tris-HCl gels as described previously (Skerker et al., 2005).
Microarray analysis of the TacA regulon
Strains were grown in PYE and harvested at OD600 = 0.4. RNA preparation was performed using the RNA-easy Kit (Qiagen). DNA arrays contained 50mer probes for each gene in the Caulobacter genome (see Table S1 for sequences). Three or more independent cultures were used for each experiment and each sample was labelled either with Cy5-dCTP or Cy3-dCTP (Amersham Biosciences). Hybridizations, scanning and data processing were performed as described previously (Laub et al., 2002) except that hybridizations contained 30% formamide and were incubated at 44°C for 6 h.
We gratefully acknowledge support from the Office of Science (BER), US Department of Energy, Grants No. DE-FG03-01ER63219 and DE-FG02-04ER63922. Support also provided in part by a National Institutes of Health grant to M.T.L. and the Bauer Center for Genomics Research at Harvard University.


text:
FIELD OF THE INVENTION
This invention relates to integrated chip diagnostics, and particulary to an improvement ABIST diagnostic method and apparatus for use in testing system components, such as SRAM and DRAM devices.
IBM is a registered trademark of International Business Machines Corporation, Armonk, N.Y., U.S.A. Other names may be registered trademarks or product names of International Business Machines Corporation or other companies.
High speed, density and reliability requirements for computer system applications of static random access memory (SRAM) require efficient and effective test applications to process and manufacture viable components for high end processor products. Manufacturing yield, and ultimately product cost is an integral part of the ability to design, manufacture, and sell computer systems in the marketplace. A key requirement to achieving leading edge technology implementations is the ability to diagnose problems in the design/manufacturing process for early resolution, in a timely fashion, and to lower overall technology cost, prior to product volume ramp-up. Efficient SRAM/DRAM designs stress the manufacturing process capability due to the high device usage (transistor elements) in a given area (smaller memory cell yields higher number of bits per chip), and the high end processor applications demand high memory bit usage. This yields an increased sensitivity to process defects over the accompanying logic, typically, and this drives the need to analyze, understand, and eventually reduce overall process defect density to achieve product yield and cost requirements.
To this end IBM uses integrated self test, and particularly for the memory arrays, the Array Built In Self Test (ABIST) has been employed to provide deterministic test coverage ensuring high quality products. Diagnosis of fails to initial manufacturing test and subsequent stress screens are critical to yield diagnostics and product reliability improvement efforts, as they are more realistic indicators than monitor structures. Monitor structures are easier to diagnose, but are only models of the product/process interaction, and are expensive from the perspective of area overhead and process productivity.
Diagnosis of array cell failures currently requires execution of the ABIST algorithm, synchronous to an external test system, during which an off-chip fail indicator is monitored, on a cycle-by-cycle basis, to determine at which points in the test the array fails. The external test equipment is used to record the failing cycles, and to repeat the test algorithms stopping “n” cycles prior to each fail, to account for the number of cycles required to “pipe” the fail indicator to an observe point, where the failing state of the array data bus can be scanned off-chip for analysis. Given the failing cycles of the ABIST algorithm, one can calculate the expected memory array data for the test and compare to the failing data scanned from the chip. The combination of the calculation of failing memory array data out, and the algorithmic calculation from the self test engine state to obtain the memory array address location together provide sufficient information for a logical cell determination of the fail. This requires clocking the array in a synchronous manner with the external test exerciser (tester) and the ability to successfully repeat failure on each of multiple passes through the test algorithm.
The technique falls short if the fails are not “hard” repeatable fails. If a fail is on the edge of the point of failure, (“soft” fails) which sometimes fails or not depending on the conditions at the time of execution, it is difficult to capture and verify. Also, AC defects (defects dependent on the speed/rate at which the algorithm is applied) are not detectable if the exerciser (tester) is not able to apply the test algorithm at the failing cycle time. This process is insufficient for diagnosis of those defects that are not detectable at test frequencies that can be provided by external test equipment, but rather rely on on-chip clock frequency multiplication techniques to multiply tester provided clock signal frequencies to those that meet or exceed the intended application.
- SUMMARY OF THE INVENTION
This process is workable for low incidents of reliability or line return fails, but is insufficient for line monitoring activity or identification of nonrandom manufacturing defects (such as mask defects, also known as “repeaters”) due to the interactive nature of the procedures, and the test hardware and manpower resources required.
This invention provides a method and apparatus for real time capture of the desired failing memory cell diagnostic information from high speed ABIST testing. This data is obtained for immediate scan-out off-chip at any level of assembly test, such as initial manufacturing wafer test, module test, including system level test, regardless of the clocking methodology. Therefore, combined with current state of the art ABIST techniques, this invention provides in-situ real time ABIST deterministic test cell fail diagnostic information. In addition, the invention provides not only for the data collection of the first failing cell, it can also be programmed to skip up to the “Nth” failing cell and record the subsequent “Nth”+1 fail instead.
The invention apparatus takes partial advantage of existing ABIST design hardware required to generate test vectors for array products, by reusing existing address register fields, and supplementing where necessary to provide for complete device under test address fields. These address registers are pipe-lined to provide data synchronous to the existing ABIST fail determination circuitry. In addition, existing ABIST comparison circuitry is used to provide a bit wise fail result vector corresponding to each of the data outs of the memory array or device under test. This fail result vector feeds a unique detect and encode circuit that determines if one and only one array data out failed, and if so, provides an encoded “address” that is concatenated to the memory array address field. Usage of the detect and encode method is proposed here as sufficient to describe failed memory array locations for efficient (minimal hardware apparatus) real time data collection.
The pipe-lined data is then fed into a hold and compare function. This hold portion of the function provides for the “full” fail address field (as defined to be the memory address of the device under test + the failing output encoded address, together sufficient to describe the failing location, or cell, in the memory array) to be stored and held in an LSSD register. The compare function provides for subsequent unique and different failing locations in the memory array to be identified. The hold and compare function is configured to load the first and reload each subsequent unique failing location encountered, decrementing the programmable skip counter at each unique fail encountered, until the skip counter reaches the final “zero” state, or the intended “Nth” fail to be recorded. It is recognized that the primary usage of the function will be to allow the default skip counter of zero thus enabling the first fail to be recorded by the aforementioned apparatus.
DESCRIPTION OF THE DRAWINGS
These and other improvements are set forth in the following detailed description. For a better understanding of the invention with advantages and features, refer to the description and to the drawings.
FIG. 1 shows a typical ABIST implementation represented by array system support elements and their configuration.
FIG. 2 illustrates another variation of an ABIST element configuration with support elements used again, but not restricted to, in support of two dimensional memory array redundancy allocation.
FIG. 3 illustrates the preferred embodiment of the ABIST diagnositic apparatus element connection for the diagnostic tests herein described
FIG. 4 illustrates that in the preferred embodiment the apparatus employed is composed of three primary elements, a fail trap register, a skip fail counter, and a hold and compare function.
- DETAILED DESCRIPTION OF THE INVENTION
Our detailed description explains the preferred embodiments of our invention, together with advantages and features, by way of example with reference to the drawings.
As we have noted, this development of our improved method and apparatus for real time capture of the desired failing memory cell diagnostic information from high speed ABIST testing uses the data obtained for immediate scan-out off-chip at any level of assembly test, such as initial manufacturing wafer test, module test, including system level test, regardless of the clocking methodology. Therefore, combined with current state of the art ABIST techniques, this provides in-situ real time ABIST deterministic test cell fail diagnostic information. In addition, the date is collected not only for the data collection of the first failing cell, it can also be programmed to skip up to the “Nth” failing cell and record the subsequent “Nth”+1 fail instead.
By virtue of the described method and apparatus we believe we have provided an extremely cost effective solution to the problem of AC (high speed) memory array cell diagnostics for both “hard” and “soft” defect mechanisms. Given that large on-chip memory macros are the primary semiconductor device yield drivers, they currently employ built in self test and some form of redundancy support for overall yield enhancement. By taking advantage of existing test infrastructure, this adds only a small percentage overhead, estimated at no more than 15% increase in overall ABIST area.
Data is collected initially and continually for immediate and continual data collection of manufacturing yield diagnostic information for every memory macro tested, whether the part yields or not, and whether or not redundant elements will be used to invoke repair. There is no test time impact, and no restriction to wafer level test data sites. Every macro, every part, every environmental test condition or test segment will have the benefit of diagnostic cell calls as part of the LSSD scan-out to obtain the manufacturing test results. See IBM Journal of Research and Development, “Boundary-Scan Design Principles for Efficient LSSD ASIC Testing ” by R. W. Bassett et al., Vol. 34, No. 2/3, March /May 1990. The use of existing array deterministic diagnostics for ABIST would typically be applied for use with a semiconductor chip which already has the circuits as described by Huott et al. in U.S. Pat. No. 5,659,441, such as a semiconductor chip having a VLSI array for storing information having redundant areas, and an array built-in, on-chip test system (ABIST) for testing the VLSI array. The existing ABIST uses data input ports, data output ports, and address ports, and includes a data control register for generating and applying deterministic data patterns to the data input ports of said VLSI array. An address control register is provided for generating addresses for application to the chip in coordination with a data control register. A comparator is provided for comparison of data inputted to the data input ports of the VLSI array from the data control register with data outputted from the data output ports of said VLSI array. This comparator includes a failing address function store for retaining failing addresses. A memory array is used for storing a plurality of microcode control vectors, each vector including a data control register field, an address control register field, a microcode pointer control register field and a read/write enable control field; a microcode pointer control register and finite state machine. There are also controls for the address control register, said data control register and said microcode pointer control register which control them in response to a plurality of microcode control vectors. There is also an overflow feedback state for the data control register and address control register. The use of redundancy provides areas for replacing normal functional areas of the VLSI array with the ABIST comparator retaining failing addresses indicating that a hardware error has been detected on power-on of said computer system element. These elements are described in detail in U.S. Pat. No. 5,659,551 “Programmable computer system element with built-in self test method and apparatus for repair during power-on”, issued Aug. 19, 1997.
In addition, for early life stress testing, burn-in, or extended voltage test, results will include diagnostic cell call information of any fail detected. This will have a significant impact by reducing traditional diagnostic re-test lead time, test equipment resource and manpower, and lead time to schedule and collect data required to guide Physical Failure Analysis efforts and establish timely problem resolution actions for line control. Subsequent level of assembly test will also see this described benefit.
The preferred embodiment of our invention will be described herein with relation to existing ABIST implementations, such as those described in patents which are mentioned below and incorporated herein by reference, including the patents of Dreibelbis et al., U.S. Pat. No. 5,961,653 entitled “Processor based BIST for an embedded memory” issued Oct. 5, 1999, and the patent of Koch et al, U.S. Pat. 5,535,164 for a BIST tester for multiple memories and will further reference this as the ABIST test engine wherein Dreibelbis describes an integrated chip having a DRAM embedded in logic is tested by an in-situ processor oriented BIST macro. The BIST is provided with two ROMS, one for storing test instructions and a second, which is scannable, that provides sequencing for the test instructions stored in the first ROM, as well as branching and looping capabilities. The BIST macro has, in addition, a redundancy allocation logic section for monitoring failures within the DRAM and for replacing failing word and/or data lines. By stacking the DRAM in 0.5 mb increments up to a 4.0 mb maximum or in 1.0 mb increments up to an 8 mb maximum, all of which are controlled and tested by the BIST macro, a customized chip design with a high level of granularity can be achieved and tailored to specific applications within a larger ASIC.
The support for two dimensional redundancy allocation as described by Hedberg and Koch specifically for memory array test is U.S. Pat. 5,859,804 entitled “Method and apparatus for real time two dimensional redundancy allocation” issued Jan. 12, 1999 which should also be and is referenced for two dimensional redundancy allocation. The U.S. Pat. 5,859,804 circuit provided an array built in self test (ABIST) system disposed on a single semiconductor chip. The chip provided a memory array having a plurality of column lines and a plurality of row lines and at least one redundant column line and at least one redundant row line with cells coupled to the lines at intersections thereof. This patent thus described a method and apparatus provided in an array built in self test (ABIST) environment formed on the semiconductor chip having an array of memory cells arranged in columns and rows and column and row redundant lines which includes testing the array along the columns to identify a given number of faulty cells in each of the columns, storing the column addresses having the given number of faulty cells in registers, further testing the array along the columns or rows to identify any additional faulty cells while masking the cells having the stored column addresses and storing the row addresses having the faulty cell in second registers until all of the second registers store row addresses, and after all of the second registers store row addresses, continue testing the array while masking the cells having the stored column or row addresses and storing the column addresses of any remaining additional faulty cell in any unused register of the first registers.
The first level of description of the preferred embodiment of the herein proposed method and apparatus are block level diagrams showing the major components of an overall ABIST system. FIG. 3 will show the appurtenant structure of the preferred embodiment.
FIG. 1 shows a typical ABIST implementation represented by array system support elements and their configuration. The redundancy allocation support described herein is, but not restricted to, two dimensional in reference to the row and column addressing within the structure of the memory array under test. There may exist a plurality of redundant row and column elements thus supported by the Redundancy Support Registers. The self test engine provides the deterministic array test patterns. The compare function has as its inputs, the expected data vector and the memory array outputs. It provides an overall pass/fail result signal which is used in combination with the gating controls from the self test engine to control the redundancy support registers in establishing real time redundancy allocation.
FIG. 2. below, shows another variation of a ABIST element configuration with support elements used again, but not restricted to, in support of two dimensional memory array redundancy allocation. The exception here, is that there may exist a plurality of row and what will be referred to as column group elements within the memory array structure. These column group elements are not selected by memory array column addressing alone, but instead coincide with a data input/output element in whole or in part.
In this art, one of the redundant/repairable element dimensions correspond to an array I/O in whole or in part. The compare element no longer compresses the test result to a single pass/fail signal, but instead provides individual result signals for each memory array data-out. The Detect/Encode circuit element is thus employed to determine memory array fail repair-ability, first by signaling that one and only one repairable element is failing and then providing an encoded representation, or “effective” address to be processed by the two dimensional redundancy support register. Again, not all memory array addressing bits may be needed by the redundant element allocation register, only those sufficient to uniquely describe/address the redundant element invocation.
FIG. 3. illustrates the preferred embodiment of the ABIST diagnositic apparatus element connection for the diagnostic tests herein described. Note shall be made that in typical support of redundancy allocation, not all memory array addressing bits may be needed. For the purposes of this embodiment, all memory addressing bits are brought to the Diagnostic apparatus. Second, the Redundancy Support Register, in this preferred embodiment, is, but not restricted to, providing two dimensional redundancy support of the memory device under test.
Thus the diagnostic register is not intrusive in existing Abist design methodologies. As can be seen, its function is parallel and independent of the remaining Abist elements. For applications different than this described preferred embodiment, additional elements of the detect and encode element can be brought out of the existing ABIST path if not needed, and used solely for the diagnostic register, slightly increasing the overhead area. In fact, this method and apparatus can be applied even when no redundancy support is required. This will in practical situations be unlikely, since the large array macros drive both redundancy applications for yield improvement, and will gain the most from yield diagnostics using this method, whereas the smaller macros typically without yield focus, will not derive as much benefit from targeted diagnostic activity.
The method and apparatus for the proposed “diagnostic register” will now be described in more detail. There are two inputs to the apparatus, namely an overall validated fail signal from the ABIST compare function, and the address buss comprised of memory array address signals concatenated with the encoded data-out address of the memory array. Any single cell fail occurring within the memory array is thereby uniquely described in a minimally encoded address. Multiple cell fails spanning more than one, or numerous, observed data bits are encoded to an all “1” s state in the encoded data out address field. The positive attributes of the encoding methodology will be discussed later in comparison to other available or potential solutions to the problem herein stated, and the potential restrictions of the methodology will be summarily resolved in light of the physical limitations of failure analysis itself.
FIG. 4 shows the ABIST diagnostic apparatus that in the preferred embodiment is composed of three primary elements, a fail trap register, a skip fail counter, and a hold and compare function. The first review will be of circuit function with the skip counter initialized to zero, or what will be referred to as “record first fail” mode, and then with non-zero values of the skip counter to “record Nth+1 fail”. “Record first fail” is considered the default or base function of the method and apparatus. The initial state of all registers is defined to be “0”, and is obtained through scan initialization of the LSSD registers in the preferred embodiment.
The skip counter output labeled “count state” is at “0” when the counter bits count down to the all “0” state or are pre-initialized to “0”, sensitizing gate a2. The address inputs are usually advancing during the course of memory testing, and will at the occurrence of any fail, by definition of including the encoded fail address of the memory array data bit, a mismatch condition will exist. Gate al is thus sensitized to the Fail Signal input pin. Given no fails (yet) from the ABIST compare during memory test, the Fail Signal input is at “0”, desensitizing gates i1 and o2, keeping gate o2 at a “1” state. If the Fail Signal input stays at “0”, then the “0” value is maintained in both the fail trap register and the hold and compare registers. Thus, as soon as the Fail Signal input goes to “1”, then the hold and compare register multiplexors will be redirected to provide the fail address as new data to the registers themselves. On the next clock event, the fail trap register will set to the “1” state. Simultaneously the hold and compare registers will set with the failing address information. Now the fail trap register controls the hold and compare multiplexors in the data hold state, such that for all subsequent clock cycles, the first fail address data is held, until the data is retrieved via the LSSD scan port of the register latches.
When the skip counter is initialized to a non zero state, then its function is to count down from the pre-initialized state, whereby its output, count state, will be “1” until the zero state of the down counter is reached. The non-zero state of the skip counter therefore blocks the fail input signal from reaching the fail trap register through gate a2. This mode is held until the intended Nth+1 fail point is reached, when the skip counter reaches the zero state, operation is as described for the above pre-initialization to “0” state. Operation of the hold and compare register is such that the address of each fail is loaded. Any subsequent fail is compared against the stored failing address, and only unique and new failing addresses will be counted by decrementing the skip counter, and reloading the new address into the hold and compare register. This is accomplished by the mismatch signal line that allows sensitization of gates al only when the new fail address is different than the last fail encountered. The hold and compare register structure is made up of an LSSD register as wide as the number of memory address and encoded address bits in total. Each register bit input is multiplexed between either the register bit itself in hold mode, or new address data when the hold signal line is “0”. The address input is connected to the multiplexor input as just described, and also one side of an exclusive-or (compare function) with the register bit itself. Therefore each bit of the entire address field is compared with the contents of the hold register on a bit by bit basis. The outputs of the exclusive-ors are logically OR'ed to form an overall mis-compare result. Multiple occurrences of the same fail in the memory array are thereby precluded from advancing the skip counter. The skip counter is implemented as a binary down counter that will advance from the preprogrammed state on each occurrence of the fail signal and a unique value of the failing address field through gate a1, and the next occurrence of a clock signal.
This method and apparatus allows for real time collection of the first failing memory array cell, and with additional initialization programming of up to the Nth+1 failing cell. This capability of in-situ collection of cell diagnostic data is provided with minimal on-chip hardware apparatus, and no impact to test time, throughput, or external test hardware resource. As was described in the problem solved description statement, this not only reduces resource and expensive re-test effort to gather this information in a traditional manner, it provides for collection of this data at levels of assembly and during high speed on product clock generation driven test that has been effectively prohibited by prior or current art to date. In addition, this data is now provided during initial wafer manufacturing final test which provides significant yield monitoring capability never provided at such low cost (or any cost) in current or prior art.
The advantage of this proposal is such that although seemingly straightforward in some ways, the unique application of a detect and encode circuit restricting encoded states to a single bit of the entire field as being the sufficient case, allows for the minimal number of states required to be encoded to be equal to the number of data-out bits +1 (multiple fail state). This represents a significant reduction in the number of hardware register bits to store effective fail information. This taken in context of today's high speed applications, the ABIST implementations utilize the same pipeline and performance techniques applied to system design. This exacerbates the hardware requirements necessary to synchronize a brute force data collection of output data from large memory array designs. The preferred embodiment described herein effectively ‘piggybacks’ onto existing pipeline data structures in place to support redundancy allocation with minimal expansion to support storage of the full memory addressing buss, but not to the pipeline depths required for the current art.
In terms of traditional diagnostic requirements that all failing data outputs need be required for diagnostic data collection, although possibly providing comfort, the information is usually not taken advantage of. This claim is made in context of physical failure analysis techniques that require a single defect to be targeted due to the investigative and destructive nature of mechanically unlayering to reach measurable and quantifiable determinants of failure mechanisms. If the defect isolation is to a peripheral circuit within the memory array causing all outputs to fail, then individual array data-out fail information is not specific, and the peripheral circuit diagnostic information is then solely available within the address field that is stored. More importantly, since it is claimed that this method and apparatus provides immediate diagnostic information for early life stress activity, then the largest number of failure mechanisms are usually singular failures of individual cells in the memory cell array. This method provides clear advantage in this area for recording the most prevalent failure mechanism.
One of the possible on-chip alternatives to the above method and apparatus which has been considered during our own development would use a straightforward pipeline of the full address buss and the full data output buss of the memory array under test up to the point of synchronization with the ABIST determination of a fail. After our analysis we determined that his alternative requires more hardware, and in some applications could result in hundreds of additional registers to store the output data necessary, to just store the first fail information, for one instance of an array macro. The method and apparatus described as our preferred embodiment has definitive benefit over this alternative.
A second alternative proposal which has been considered for resolving the requirements of diagnostic data collection was to provide an additional copy of the memory array macro on-chip, and to reconfigure ABIST to test the target memory while storing the bit by bit compare results into the “spare” memory macro. Then at some point, another macro becomes the target, and the previously successfully tested target becomes the new data collection/storage device. This second alternative proposal is theoretically viable but we believe it would add additional complexity and area impacts on chip as compared to the preferred embodiment.
A third alternative to the preferred embodiment which has been considered is a alternative which would provide extensive on product clock generation control circuitry to the chip to be tested, whereby the effective number of on product generated clock cycles can be counted and specifically controlled such that a more traditional approach to diagnostic data collection can be implemented. We believe that this is also a valid and workable approach, even though it is not our preferred embodiment because when such circuitry resides on chip for purposes of system control and it can also be used for memory array cell diagnostic activity, although again we do not consider this our preferred embodiment because it does involve more tedious and interactive data collection algorithms to achieve comparable results. If this circuitry, design complexity, and intrusion into the basic clocking methodology of the chip infrastructure is required for test support, then, the design and usage resource requirements (both hardware and manpower) will normally preclude the design benefit obtained by the third alternative.
The application of our method and appratus is particularly useful in system devices related to high speed processing of data, and can be implemented in devices such as used for cache memory ABIST for high speed processors, including double processor chips where elements are divided into two smaller chips which can be separately tested for improvements in yield and cost, as well as for other memory units and array designs used in complex logic. The design can enable improvements in implementation of complex logic designs allowing CMOS performance to improve with merged logic-DRAM, SDRAM, low power technologies, and unique packaging alternatives, and enable processor speed and cost improvements in addition to those obtained by cycle time improvements due to technology scaling.
While the preferred embodiment to the invention has been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.

domain: design


text:
If you're a fan of eBoy's pixel-tastic work you should check out this little video. The film documents the process for 4 illustration pieces based on Irish cities. All created during a recent campaign for Coca-Cola by McCann Erickson in Ireland.
It's always good to get a glimpse into the craft of other illustrators and creatives. Cheers to The Small Print & Scamp.ie for the heads up.

domain: hobby


A-B-C Factory-trained Field Service Technicians
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text:
Official Memorandum Issued 11/05/2010
November 5, 2010
Dear County and District Superintendents, Special Education Local Plan Area Directors, Special Education Administrators at County Offices, Charter School Administrators, Principals, and Nonpublic School Directors:
UPDATE: ENSURING THE CONTINUOUS DELIVERY OF MENTAL HEALTH SERVICES TO STUDENTS WITH DISABILITIES
You may be aware that the Federal United States District Court for the Central District of California issued a Temporary Restraining Order (TRO) [Case No. 2-CV-10-07956-GW (AGRx)] on November 1, 2010. This TRO requires Los Angeles County Office of Education (LACOE) to disburse its share of the statewide $76 million in federal funds to Los Angeles County Department of Mental Health (LACDMH) for the purpose of providing educationally-related mental health assessments and services to eligible special education students upon referrals by local educational agencies. Further, the TRO specifies that these funds must be used according to the interagency agreement that existed between LACOE and LACDMH prior to the Governor’s October 8, 2010 veto of the 2010–11 appropriation for educationally-related mental health services mandated by Assembly Bill (AB) 3632.
In keeping with how funds have been dispersed in the past, the California Department of Education (CDE) prepared and sent Grant Award Notifications to County Offices of Education that allocated $76 million in federal funds to contract, on behalf of Special Education Local Plan Areas (SELPA) in their counties, with the appropriate mental health agency to provide specified mental health services. We anticipate that these funds will provide urgent relief to ensure that students receive needed services via existing interagency agreements in accordance with state and federal law [Chapter 26.5 of Division 7 of the California Government Code sections 7570-7590, 34 Code of Federal Regulations (CFR) 300.704(b)(4)(iii)] .
The TRO gives substance to the CDE’s belief that county mental health (CMH) agencies remain responsible for carrying out their requirements to serve students with disabilities who are eligible to receive services under AB 3632 provisions and your local interagency agreements. In my previous communication with you, I provided the relevant legal citations for your reference. I also informed you that the CDE is contemplating serious action, up to and including all available administrative and judicial actions, if necessary.
The CDE’s primary interest and responsibility is to ensure that the special education and related services documented in a child's individualized education program (IEP) are provided by the responsible parties. Continuation of services is vital while the specifics of funding are resolved either through the courts or through legislative action.
I encourage you to continue making appropriate student referrals to your local CMH agency. The CMH agency is required to then determine whether the referral is appropriate and, if so, to complete the assessment, develop a written report, review the report at an IEP meeting, and make a recommendation about whether the student requires mental health services. If the CMH agency representative recommends that the student receive AB 3632 services then by agreement of the student's IEP team, the services are included in the student's IEP. The services cannot be removed from the IEP without mutual agreement of the CMH agency, the local educational agency (LEA), and the student's parents or guardians, or by judicial order.
Many CMH agencies have continued to operate in a manner that comports with their requirements under various federal and state statutes. However, some CMH agencies have notified LEAs that they will not continue to provide their required services because they believe that the suspension of the mandate is valid or because the Governor’s veto of the specific funding earmarked for mental health services makes it difficult for them to do so. The CDE will continue to investigate and enforce corrective actions for any violations of state and federal laws and regulations pertaining to students with disabilities.
There are several potential courses of action for LEAs should CMH agencies decline to meet their responsibilities. The proper course of action in your particular instance should be discussed with your legal counsel. Pending all actions, LEAs must continue to refer appropriate students to CMH agencies, and if students are currently receiving CMH services, LEAs must ensure that students continue to receive all IEP services that were being provided by CMH agencies before the Governor’s veto. Such services include counseling and paying for the cost of a residential placement.
LEAs should follow the interagency dispute resolution process as mandated in Government Code Section 7585 et. seq. when CMH agencies refuse to provide IEP-related AB 3632 services. This process requires the LEA to submit a written complaint to the Superintendent of Public Instruction and/or the Secretary of the California Health and Human Services Agency. The complaint should allege the failure of the CMH agency to provide a service to a specific student. This process could be used, for instance, if the CMH agency is continuing to provide mental health services to a student, but is not sending representatives to the student's IEP meeting. The interagency dispute resolution process is also appropriate if the CMH agency refuses to attend an IEP meeting to discuss placing a student in a residential placement. Before LEAs file an action in court against a CMH agency, the LEA must first avail itself of its administrative remedies.
I advise LEAs to inform their student's parents and guardians that although the LEA cannot control the actions of a CMH agency, parents and guardians have the right to file a request for a due process hearing against the CMH agency. In addition, a student's parents or guardians may file a special education due process complaint and name either or both the CMH agency and the LEA as parties to the complaint.
The CDE representatives will continue to meet with legislative staff to determine a resolution to the veto of the state funding for these important services, and we will provide updated information to you as it becomes available.
If you have any general questions regarding this subject while we work toward a more permanent solution, please contact the Special Education Division by email at email@example.com.
Original signed by Fred Balcom. Hard copy of the signed document is available by contacting the Special Education Division's Director's Office at 916-445-4602.
Fred Balcom, Director
Special Education Division

domain: law