Fast uncoiling kinetics of F1C pili expressed by uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> are revealed on a single pilus level using force-measuring optical tweezers

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S_I to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400–700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.     

Inheritance of leaf peroxidase isoenzymes in Nicotiana alata and linkage with the S-incompatibility locus

Summary Genetic analysis of peroxidase isoenzymes observed by electrophoresis shows that each of the two cathodic bands are controlled by one gene, respectively, P_I and P_II. Each gene has two allele forms; presence of activity (dominant) and absence of activity (recessive). The same situation is found for one anodic band; the three other anodic bands are controlled by a single gene with three active allele forms. No progenies seem to be produced from gametes P _I ^- P _II ^- (no activity of P_I or P_II). Investigation of the incompatibility system and the isoperoxidases demonstrates that the loci P_I, P_II and S are located in the same chromosome. P_I is closely linked to the S locus (3 cM); the distance between P_II and the S locus is 34 cM.     

Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

 CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, Cu_A site in nitrous oxide reductase were obtained after insertion of ⁶⁵Cu or both ⁶⁵Cu and ¹⁵N-histidine. The ¹⁴N/¹⁵N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for ¹⁴N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to Cu_A, indicating that the ground state is ²B_3u. Proton hyperfine structure was detected from four C_β protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of Cu_A. The [N_HisCu(RS)₂CuN_His]⁺ core structure of Cu_A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined Cu_A* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the Cu_A structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of Cu_A is best represented by the resonance structures of the form [Cu^IS^–S^–Cu^II↔ Cu^IS^•S^–Cu^I↔ Cu^IS^–S^•Cu^I↔ Cu^IIS^–S^–Cu^I]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster. Received: 17 September 1997 / Accepted: 28 October 1997     

Molecular analysis and mapping of two genes encoding maize glutathione S-transferases (GST I and GST II)

Maize glutathione S-transferase (GST) isozymes are encoded by a gene family comprising at least five genes, three of which (Gst I, II andIII) have recently been isolated and sequenced. The enzymes are active as homo or heterodimers and exhibit intraspecific polymorphism including a “null” variant for the two major isoforms expressed in roots. Northern blot analyses performed on total root RNA from “null” and “plus” genotypes, usingGst I- andGst II-specific probes, indicated that theGst I gene controls the expression of the two major GST isoforms expressed in roots.Gst I andGst II were mapped by RFLP analysis using an F₂ population of 149 individuals previously characterized.Gst I was localized on the long arm of chromosome 8, while two putativeGst II loci were mapped to chromosomes 8 (70 cM fromGst I) and 10, respectively.     

The Intracellular Citrus Huanglongbing Bacterium, ‘Candidatus Liberibacter asiaticus’ Encodes Two Novel Autotransporters

Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘ Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasA_I and LasA_II in lieu of the previous names Hyv_I and Hyv_II. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasA_I and LasA_II share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasA_I C-terminus and the full length LasA_II were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasA_I was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasA_I protein. Its surface localization was also confirmed by the removal of the LasA_I antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasA_I translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.     

RNase X2, a pistil-specific ribonuclease from Petunia inflata,shares sequence similarity with solanaceous S proteins

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S₃ and S₆ proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001     

Properties of placental alkaline phosphatase. I. Molecular size and electrical charge of the various electrophoretic components of the six common phenotypes

The properties of the six common phenotypes (F₁, S₁, I₁, F₁S₁, S₁I₁, F₁I₁) of placental alkaline phosphatase have been studied by DEAE-Sephadex chromatography, gel filtration, two-dimensional paper-starch gel and starch block electrophoresis. The rapidly migrating component (component I) had the same molecular weight (118,000) in all phenotypes studied but different negative charge (F₁>I₁>S₁). The fastest of the slow-moving components (component II) had a molecular weight of 130,000 and a higher absolute negative charge, but slightly lower net negative charge than I. The very slow-moving components (II) were usually composed of two major and several minor components. The major components had a higher absolute negative charge but a lower net negative charge than II. All II components had a molecular weight of over 200,000. It is suggested that II is an enzyme bound to a small molecule, while II components are aggregates of enzyme with various substances or with itself.This work was supported in part by U.S. Public Health Service Grant HD02552. K.H. is a Career Scientist of the Health Research Council of the City of New York (I-513).     

Oligomycins A and C,major secondary metabolites isolated from the newly isolated strain <Emphasis Type="Italic">Streptomyces diastaticus</Emphasis>

During the screening program for fungicides, one actinomycete strain ECO 00047 was isolated with the potential activity against fungus. According to the morphology and analysis of the nucleotide sequence of the 16S rRNA gene (1500 bp) this isolate was identified as Streptomyces diastaticus. The active compounds were separated by silica gel column chromatography, Sephadex LH-20 gel filtration and then purified by flash chromatography on C₁₈ (20–45 μm). The chemical structure of the bioactive compounds I and II were elucidated, based on the spectroscopic data of MS, IR, UV, ¹H-NMR, ¹³C-NMR and X-ray single crystal diffraction analysis. Compounds I and II were identical with oligomycins A and C, the macrolide antibiotics which have been known to be produced by Streptomyces diastatochromogenes, S. libani and S. avermitilis. The two compounds exhibited a strong activity against Aspergillus niger, Alternaria alternata, Botrytis cinerea and Phytophthora capsici but no activity toward bacteria. Although the two above antibiotics were known, their isolation has so far not been reported from S. diastaticus.     

Functional organization of auditory projections to somatosensory cortical areas in cats

The functional properties of fibers transmitting auditory impulses to somatosensory areas S_I and S_II were studied in anesthetized and waking animals by the evoked potentials method. The thresholds of evoked potentials in areas S_I and S_II are 15–35 dB higher than those of evoked potentials in the auditory projection areas. Tonotopical localization is absent in somatic areas. Experiments on anesthetized animals showed that the spread of impulses relating to acoustic stimuli of different frequencies into areas S_I and S_II is effected through area A_I and its connections with the above zones. Another pathway probably also participates in the conduction of impulses from clicks. Analysis of the time constants of the first positive potential suggested that the interneuronal organization of auditory projections to area A_I is less complex than that of projections to the somatosensory areas. Comparison of amplitudes of evoked potentials of different projection zones in area S_I showed that the projection of the head receives more auditory impulses than the projection zone of the forelimbs, confirming the morphological data published previously.     

Derepressed plasmids of incompatibility group I1 determine two different morphological forms of pilus

IncI₁ plasmids R144drd3 and R64drd11 determined thick, probably rigid pili in addition to the well-known thinner I₁ pili. Electron microscopy revealed that the thick I₁ pili often had large knobs at one end and were pointed at the other. They did not appear to act as receptors for any I-specific bacteriophages.     

A structural basis for sustained bacterial adhesion: biomechanical properties of CFA/I pili

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.     

Induction of isoenzymes of alcohol dehydrogenase in “flor” yeast

Summary Two isoenzymes of alcohol dehydrogenase (adh I and adh II) from Saccharomyces cheresiensis have been differentiated by thermal treatment of the crude extracts. The effect of pH on the stability and the K _m for ethanol are different for the two isoenzymes.The proportions in which they are present depend on the carbon source used by the yeast: adh I is the major component in cells grown on glucose, and adh II in those grown on ethanol. Cells grown on glucose plus ethanol show high levels of both isoenzymes, indicating that the synthesis of adh I is subjected to nutritional induction by glucose, and that of adh II by ethanol.The physiological roles of the two isoenzymes are discussed in relation with the nutritional characteristics of S. cheresiensis.     

Comparison of Soybean Tissue Extracted with Different Solvents

The enantioselective hydrolysis of (R,S)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4-(3,5-dinitrobenzoyl)-4H-[1,4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)- and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (III), for which the optical purity was estimated to be >96%e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.     

Amino acid composition of fractions of ‘Kentucky-31’ tall fescue as affected by N fertilization and mild water stress

Summary Environmental and management factors can influence the protein concentration of forages, significantly altering specific amino acid content. Drought, high rates of fertilizer N and the presence of a fungal endophyte have been associated with significant alterations in plant N metabolites and animal performance problems on tall fescue. A controlled environment study was conducted to examine the influence of N fertilization (10 and 100 gN/g) and water regime (low and adequate soil water availability) upon the distribution and concentration of amino acids in endophyte infected tall fescue (Festuca arundinacea, Schreb.) herbage. Tall fescue tissue was collected from three replicates of each treatment, quick frozen in liquid N and lyophilized. Two insoluble (R_I, structural residue; R_II, membrane residue) and two soluble (S_I, soluble protein; S_II, low molecular weight N compounds) fractions were collected. Amino acid analyses of acid hydrolysates of fractions showed that application of 100 N significantly increased the concentration (per unit dry weight) of all amino acids in the entire plant, with an average increase of about 55%. Application of 110 N increased the concentrations of most amino acids in fractions R_I, R_II, and S_I, but only aspartate-asparagine, glutamate-glutamine, alanine, threonine, serine, valine and proline in fraction S_II. Fraction R_I contained about 65% of total amino acids under 10 N and 55% under 110 N even though N level did not alter dry matter distribution among fractions. While the amount of dry matter was least in S_I, amino acids in the fraction ranged from 8% (leucine, 10 N) to 20% (lysine, 110 N) of the total amount of specific amino acids recovered. Significant increases in proline, glutamate, aspartate, serine, valine, threonine, alanine and phenylalanine concentration occurred under low soil-water availability compared with adequate water conditions. Basic amino acids including histidine, arginine and lysine increased with increased N and with water stress at each N level. Application of N increased amounts, and water stress influenced distribution of amino acids among the fractions of tall fescue herhage. Nitrogenous components, such as non-protein amino acids which could influence plant nutritive quality, were increased in fraction S_II by increased N and water stress.     

Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.     

The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment

Uropathogenic Escherichia coli express pili that mediate binding to host tissue cells. We demonstrate with in situ force measuring optical tweezers that the ability of P and type 1 pili to elongate by unfolding under exposure to stress is a shared property with some differences. The unfolding force of the quaternary structures under equilibrium conditions is similar, 28 +/- 2 and 30 +/- 2 pN for P pili and type 1 pili, respectively. However, type 1 pili are found to be more rigid than P pili through their stronger layer-to-layer bonds. It was found that type 1 pili enter a dynamic regime at elongation speeds of 6 nm/s, compared to 400 nm/s for P pili; i.e., it responds faster to an external force. This possibly helps type 1 to withstand the irregular urine flow in the urethra as compared to the more constant urine flow in the upper urinary tract. Also, it was found that type 1 pili refold during retraction at two different levels that possibly could be related to several possible configurations. Our findings highlight functions that are believed to be of importance for the bacterial ability to sustain a basic antimicrobial mechanism of the host and for bacterial colonization.     

Two differentially regulated nitrate reductases required for nitrate-dependent,microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O₂, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NR_I) and 200 kDa (NR_II). NR_I and NR_II were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO₃. However, only NR_I was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O₂ tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NR_I, while the other two mutants, GRF3 and GRF4, expressed mostly NR_II. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NR_I) or by both microaerobiosis and nitrate (NR_II).Abbreviations NR Nitrate reductase - M _r Relative molecular mass - PMSF Phenylmethylsulfonyl fluoride     

The Conformer Substates of Nonoriented B-type DNA in Double Helical Poly(dG-dC)

Abstract

A nonoriented hydrated film of poly(dG-dC) with ≈20 water molecules per nucleotide (called B by Loprete and Hartman (Biochem. 32, 4077–4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)₂ dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on B_Ito-B_II interconversion in the classical B-DNA forms are very similar to those in the B-form, and that the spectroscopic differences between the B_I and B_II features from classical B-DNA and those from the modified B-form are minor. Nonexponential kinetics of the B_I→B_II transition in the B-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (≈30 versus ≈10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is B_II→B_I transition on cooling from 290 K and B_I→B_II transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263–3274 (2002)).     

A new class of clear mutants from coliphage 434 hy not complementing CII and C3 mutants for lysogenization

Summary Clear mutants which differ from regular C _I , C _II , CIIIand y mutants have been isolated from phage 434 hy. These mutants resemble C _I mutants in plaque and spot phenotype but efficiently complement C _I mutants for lysogenization. Like C _II mutants, they do not complement authentic C _II mutants for lysogenization but in contrast to C _II mutants they also fail to complement C _III mutants. They map between the lambda-434 non-homology region and Co ₁ (aC _II mutant). On account of this map position adjacent to C _II the mutants of the new type are called C _IIa . They arise from phage 434 hy with a frequency comparable to that of C _I and C _II mutants. Such mutants are also obtained from phage lambda but apparently not from phage b₅. C _IIa mutants would not fit into a picture of three independently acting cistrons C _I , C_II, and C _III . The hypothesis is presented that C _IIa and C _II mutants are in the same structural gene. Two possibilities are discussed that would account for the complementation patterns: 1. C _IIa mutants may block the expression of gene C _III in cis position; or 2. the products of genes C _II and C _III function through an oligomeric complex they form.