Ribosomal proteins. Most RP genes of ≥100 codons (28 of 36) are PHX and all have E(g) ≥ 0.93 (marginally PHX). Interestingly, RP genes in Synechocystis do not reach top expression levels, as occurs for most eubacterial genomes (4). In fact, of 17 complete eubacterial genomes analyzed only Synechocystis and Thermotoga maritima have no RP genes among the highest 10 PHX genes. The RP gene with the highest PHX value in Synechocystis is rpS2; this gene attains E(g) = 1.30 (rank 43). The RP genes are rarely duplicated in bacterial genomes, but Synechocystis contains two copies of the S1 RP. Both of the rpS1 genes encode proteins of reduced lengths (328 and 305 amino acids) compared to the eubacterial S1 length, which generally exceeds 500 amino acids (4).

Translation/transcription processing factors (Table 2). Factors primary for translation/transcription typically carry the highest E(g) values. For example, the elongation factor EF-G attains the highest E(g) value in the genomes of Haemophilus influenzae, B.subtilis, Helicobacter pylori, Rickettsia prowazekii, Chlamydia trachomatis and Pyrococcus abyssi (4). In Synechocystis, EF-G (fus) is encoded by four similar genes of E(g) values between 1.43 and 0.68 (Tables ​(Tables22 and ​and66).

Chaperones (Table 3). This group includes genes encoding the principal chaperones (e.g. heat shock proteins groEL, dnaK, grpE and htpG), as well as proteins involved in trafficking, secretion and protein degradation. Many genes in this category are PHX and several are present in multiple copies. For example, there are three homologs of dnaK. The homolog that registers the lowest E(g) value, sll0058, may be specifically involved in pilus biogenesis (D.Bhaya, A.Takahashi and A.R.Grossman, unpublished results) and therefore may not be required at as high a level as the more commonly used chaperones. In contrast, both GroEL homologs are among the top PHX genes (Table ​(Table3).3). Of 17 complete eubacterial genomes analyzed (4) Synechocystis, Mycobacterium tuberculosis and Vibrio cholerae carry two copies of the groEL genes, but only Synechocystis maintains both copies as PHX.

Aminoacyl tRNA synthetases and modification genes. Among genes functioning in translation, RPs and translation processing factors tend to be PHX while aminoacyl tRNA synthetases are generally not PHX (except in Escherichia coli; S.Karlin, J.Mrázek, A.M.Campbell and A.D.Kaiser, submitted for publication). The genes encoding S-adenosylmethionine synthetase (metX) and aspartyl-tRNA synthetase (aspS) are PHX in Synechocystis, although it is not clear whether or not to place metX in this functional class since S-adenosylmethionine participates in a number of different cellular processes (14).

Photosynthesis and respiration (Table 4). Most of the genes encoding proteins that participate in photosynthesis and respiration are PHX. The macromolecular complexes essential for photosynthesis are PSI and PSII, the light-harvesting complex or PBS, the cytochrome b₆f complex and ATP synthase. Almost all components of these complexes are PHX or marginally PHX [E(g) ≥ 0.95] and are often among the PHX genes with the highest E(g) values (Tables ​(Tables11 and ​and44).

Of the 23 genes directly involved in CO₂ fixation, 13 are PHX. Several of the genes in this class having the highest E(g) values also function in glycolysis; these include fructose bisphosphate aldolase (fda and cbbA), glyceraldehyde 3-phosphate dehydrogenase (gap) and phosphoglycerate kinase (pgk). Enigmatically, another key enzyme functioning in both glycolysis and photosynthetic CO₂ fixation, triosephosphate isomerase (tpi, slr0783), is not PHX [E(g) = 0.77]. Between the two copies of gap genes, gap2 [E(g) = 1.30] encodes a protein that appears to function in both CO₂ fixation and glycolysis and is expressed at high levels under diverse conditions, whereas expression of gap1 is hardly detected (15), even though it is PHX [E(g) = 1.07]. Ribulose 1,5-bisphosphate carboxylase (Rubisco) catalyzes the carboxylation of d-ribulose 1,5-bisphosphate, which participates in the primary reaction of CO₂ fixation. Genes encoding the large and small subunits of RuBP carboxylase, rbcL and rbcS, are both PHX [E(g) = 1.33 and 1.13]. Synechocystis has five genes encoding homologs of the CO₂-concentrating mechanism protein CcmK. CcmK may be involved in formation of the carboxysome, the site of RuBP carboxylase sequestration. Two of the five genes are PHX [sll1028 and sll1029, both with E(g) = 1.20], while the other three are not PHX (slr1838, slr1839 and slr0436). Interestingly, sll1028 (110 amino acids) and sll1029 (102 amino acids), as well as sll1838 and sll1839, are tandemly arranged, whereas slr0436 may represent a fusion of two ccmK homologs to generate a single ORF of 296 amino acids.

Among the genes for electron carriers that function in photosynthesis and/or respiration, the main PHX gene encodes plastocyanin (PetE) [E(g) = 1.32]. The Synechocystis genome contains a single copy of the petE gene (sll0199). In contrast, another electron carrier essential in photosynthesis, ferredoxin, is encoded by four homologous genes. Multiple ferredoxins may be important for guiding electron flow to specific electron acceptors; electrons from ferredoxin can be used to reduce NADP (and CO₂), nitrite or sulfate. Two of the genes encoding ferredoxin register as PHX and, notably, the ferredoxin gene slr0150 is PA.

Cytochrome c oxidase, composed of the three subunits CtaC, CtaD and CtaE, is a complex of the respiratory chain that catalyzes the reduction of oxygen to water and generates an electrochemical potential that can provide energy for numerous cellular processes. There are two cytochrome c oxidase operons on the Synechocystis PCC6803 genome. The first operon at position 790–794 kb includes all three cta genes, while the second operon at position 1540–1542 kb contains ctaD and ctaE, with ctaC located at 1698 kb. The latter genes are not PHX.

Energy metabolism (apart from photosynthesis and respiration). Seven glycolysis genes typical of most bacteria are PHX. These genes also function in photosynthetic CO₂ fixation, which apparently requires high level expression. None of the genes encoding TCA cycle enzymes are PHX in Synechocystis, which contrasts with the finding that several TCA cycle genes from most eubacteria are PHX (4). However, cyanobacteria maintain a truncated TCA cycle (16,17) and the major catabolic pathway for supplying cells with energy and reductant for respiration, dark growth and nitrogen fixation is the oxidative pentose phosphate pathway (18). Therefore, unlike most bacteria, cyanobacteria may not require high expression levels of TCA cycle enzymes and indeed these enzymes are not highly expressed. Among the oxidative pentose phosphate pathway enzymes, 6-phosphogluconate dehydrogenase (Gnd) and phosphogluconolactonase (DevB) are marginally highly expressed [E(g) = 1.00 and 0.96, respectively], whereas the first enzyme of the pathway, glucose 6-phosphate dehydrogenase (Zwf), records a reduced E(g) of 0.77. The essential enzymes of the non-oxidative part of the pentose phosphate pathway, transketolase and transaldolase, are both PHX at the levels E(g) = 1.31 and 1.21, respectively.

Replication and repair. In most bacteria and in Synechocystis, genes functioning in DNA replication are generally not PHX. The replication initiation protein DnaA is marginally PHX [E(g) = 1.05] and one of two genes encoding DNA gyrase subunit A (slr0417) is PHX [E(g) = 1.18]. As verified in many other prokaryotic genomes, the genes encoding the single-stranded DNA-binding proteins Ssb and RecA, which function in repair and replication, qualify as PHX.

Regulatory proteins. Bacterial genes encoding regulatory proteins are seldom PHX. In Synechocystis PCC6803, 13 of 133 putative regulatory genes are PHX, but all have E(g) < 1.12. Four regulatory proteins satisfy Definition II as PA genes, including sll0709 (LlaI.2, required for the LlaI restriction system), sll1408 (regulatory protein PcrR), sll0776 (eukaryotic protein kinase PknA) and sll0797 (a regulatory component of the sensory transduction system OmpR subfamily). Some of the regulatory elements may be toxic to bacterial cells when expressed above a threshold level. The PA character of a gene could result in low levels of expression, which would diminish the potential of the gene product to become toxic to the cell. With respect to LlaI.2, restriction enzymes are often laterally transferred among bacterial strains and may be recognized as PA in the recipient bacterium (9,19).

PA genes form three major clusters and several smaller clusters. A cluster covering positions 353–385 kb contains the genes rfbF, rfbE, rfbU and galE1, one transposase (slr1075) and 13 ORFs of unknown function. The rfb genes may be required for lipopolysaccharide biosynthesis. Lipopolysaccharide biosynthesis genes of bacteria are often alien, as noted for E.coli (21). Three ORFs in the cluster (slr1063, slr1065 and slr1066) show weak similarity to glycosyl and galactosyl transferases, which function in lipopolysaccharide biosynthesis, and slr1616 is weakly similar to GalE.

For a number of protein complexes, the predicted expression levels E(g) of the genes encoding the polypeptides of the complex do reflect to some extent subunit stoichiometry. For instance, the catalytic sector of ATP synthase (CF₁) consists of five subunits with a stoichiometry α₃β₃γ₁δ₁₁. As expected from this holoenzyme composition, the genes encoding the α and β subunits have higher predicted expression levels than the genes encoding the other three subunits. In the ATP synthase membrane sector (CF₀), which functions as a transmembrane proton channel, the only subunit encoded by a gene that qualifies as PHX is present in CF₀ at 6–12 copies; the non-PHX subunits are present in CF₀ as single copies (Table ​(Table4).4). Interestingly, genes encoding the subunits of CF₁ exhibit higher predicted expression levels than genes encoding CF₀ subunits. We speculate that the extrinsically located CF₁ sector of ATP synthase is exposed to potentially damaging agents resulting in higher turnover rates, which would necessitate a more rapid synthesis of CF₁ subunits than CF₀ subunits; CF₀ subunits may be partially protected by sequestration in the membrane. Notably, all of these genes are clustered on the Synechocystis genome at two sites (atpB and atpE at one site and atpI, atpH, atpG, atpF, atpD, atpA and atpC at the other). The expression levels of the genes in these clusters may be dictated in part by different rates of transcription, differential stabilities of the mRNAs and segmental stability differences in polycistronic messages.

The heterodimers phycocyanin and allophycocyanin are abundant proteins associated with PBSs, the major light-harvesting complex in cyanobacterial cells. There is an excellent correspondence between the PHX character of genes encoding the α and β subunits of the individual phycobiliproteins (cpcBA for α and β phycocyanin and apcAB for α and β allophycocyanin) (Table ​(Table4)4) and the abundance of these polypeptides in the cell. However, the highest predicted expression level, E(g) = 1.51, of a PBS polypeptide is for the core-associated membrane linker ApcE. This is surprising given the stoichiometry of the individual polypeptides in the PBS. The PBS is a macromolecular complex of more than 15 polypeptides that together can constitute >30% of all cellular proteins (3,22). Only two copies of ApcE are required per PBS, while many of the other PBS components are more abundant. For example, a PBS may contain 18 subunits each of ApcA and ApcB and variable numbers (generally between 36 and 108) of the CpcA and CpcB subunits. The phycocyanin-associated linker, CpcC, is present at six or fewer copies per PBS, depending on light conditions (23). Moreover, there is no evidence that the rate of ApcE turnover is high relative to that of other subunits of the PBS, although it appears to be more labile during isolation of the PBS. A possible cause for the high E(g) value of the apcE gene may relate to its size. The apcE gene is 895 codons in length whereas all other PBS components are <300 codons. It has been observed in E.coli that codon choices in long genes are more biased than in short genes (24). An analogous situation arises with genes encoding the PsaA and PsaB subunits, which have very high PHX values [E(g) = 1.40 and 1.41, respectively] but are present as one copy each per photosystem I. However, like ApcE, they are very high molecular mass polypeptides. These results raise the possibility that quantitative estimates of the expression levels E(g) may not be fully comparable for genes of very different lengths and that expression levels of genes encoding large proteins may be overestimated by this method.

The PSII core polypeptide D1 (PsbA) is encoded by three highly homologous genes, psbA1, psbA2 and psbA3, in Synechocystis. All three genes encode polypeptides of identical lengths. While the PsbA2 and PsbA3 polypeptides have identical amino acid sequences, the sequence of PsbA1 is ~80% identical to that of PsbA2 and PsbA3. Codon usages are very similar among these genes, yielding predicted expression levels E(g) of 1.27, 1.23 and 1.23 for psbA1, psbA2 and psbA3, respectively. The high sequence similarity and the codon bias typical of highly expressed genes may reflect the establishment of this gene family through recent gene duplications. The measured levels of the individual psbA mRNAs are strikingly different; psbA2 accounts for >90% of cellular psbA mRNA, psbA3 for <10% and psbA1 is cryptic (25), suggesting that transcription of the genes is differentially regulated. This is in accordance with the finding that the three genes have highly diverged 5′-promoter regions. Interestingly, the cryptic psbA1 gene produces a fully functional protein when fused with the 5′-region of psbA2 (26). It is enigmatic that a cryptic gene such as psbA1 would retain a high E(g) value and produce a functional protein unless there are specific environmental conditions (that have not been identified) that trigger transcription of the gene.