The Next Step: Formulating State Machine Models for Microbes:
by Ross Overbeek
October, 2010

1. First, I will go through some examples of conjectures that follow naturally from a minimal study of atomic regulons (sets of genes that are always co-expressed). My goal is to show that a very simple integration of data (relating to operons, subsystems, and expression data) produces truly impressive results. I have recently collected well over a hundred examples of atomic regulons that can be easily used to produce testable conjectures.
2. Then, I will reflect on why these conjectures are easy to produce.
3. I will argue that they are just a by-product of a more important issue -- the need to construct state-machine models based on atomic regulons.
4. Finally, I will describe what I see as the pipeline needed to really exploit atomic regulons.

Some Examples in the Use of Atomic Regulons

An Example Relating to Ubiquinone/Menaquinone Metablism

Pegs in Atomic Regulon 14 [ON=888 OFF=6]

1. First, let us note the expression profiles. It appears that each of the clusters was expressed in 888 experiments and not expressed in 6. It would be good to know what those six experiments were, I think.
2. The fact that significant genes from the same metabolic activity (ubiquinone/menaquinone metabolism) are present in both clusters supports the position that the clusters are actually related.
3. It needs to be noted that, while the first cluster may be an operon, the second is not. The gene fig|83333.1.peg.3768 is a transcriptional regulator that breaks the cluster into two potential operons (each containing two of the ubiquinone/menaquinone genes).

• The PEG fig|83333.1.peg.3761 is embedded between ubiquinone/menaquinone gene in a single operon. It is clearly a good candidate for a role in ubiquinone/menaquinone metabolism.
• A similar comment could be made for the tatABC genes. The Tat (twin-arginine translocation) system mediates export of periplasmic proteins in folded conformation. It would take an expert to understand how this might relate to the metabolism in question, if at all. However, it does seem to me that both processes (the electron transport and movement of proteins across a membrane) might be related somehow.

  The Twin-Arginine Transport System
  
  Frank Sargent, Ben C. Berks and Tracy Palmer
  
  The twin-arginine transport (Tat) system is a protein-targeting
  pathway found in the cytoplasmic membranes of many eubacteria, some
  archaea, and the chloroplasts and mitochondria of plants. It is
  apparently not a feature of animal physiology. Substrate proteins are
  targeted to a membrane-bound transport apparatus by N-terminal signal
  peptides harbouring a distinctive ‘twin-arginine’ amino acid sequence
  motif, and, most remarkably, all substrate proteins are transported in
  a fully folded conformation. Model systems most commonly used to study
  the fundamentals of Tat transport are the Gram-negative eubacterium
  Escherichia coli, the Gram-positive eubacterium Bacillus subtilis, and
  thylakoid membranes derived from pea or maize chloroplasts.
  
  

• The RmuC protein is not terribly well characterized. The sole paper that I could find was

  Genes Cells. 2000 Jun;5(6):425-37.
  
  Genes involved in the determination of the rate of inversions at short inverted repeats.
  
  Slupska MM, Chiang JH, Luther WM, Stewart JL, Amii L, Conrad A, Miller JH.
  
  Abstract
  
  BACKGROUND: Not all of the enzymatic pathways involved in genetic
  rearrangements have been elucidated. While some rearrangements occur
  by recombination at areas of high homology, others are mediated by
  short, often interrupted homologies. We have previously constructed an
  Escherichia coli strain that allows us to examine inversions at
  microhomologies, and have shown that inversions can occur at short
  inverted repeats in a recB,C-dependent fashion.
  
  RESULTS: Here, we report on the use of this strain to define genetic
  loci involved in limiting rearrangements on an F' plasmid carrying the
  lac genes. Employing mini-Tn10 derivatives to generate insertions near
  or into genes of interest, we detected three loci (rmuA,B,C) that,
  when mutated, increase inversions. We have mapped, cloned and
  sequenced these mutator loci. In one case, inactivation of the sbcC
  gene leads to an increase in rearrangements, and in another,
  insertions near the recE gene lead to an even larger increase. The
  third gene involved in limiting inversions, rmuC, has been mapped at
  86 min on the E. coli chromosome and encodes a protein of unknown
  function with a limited homology to myosins, and some of the SMC
  (structural maintenance of chromosomes) proteins.
  
  CONCLUSIONS: This work presents the first example of an anti-mutator
  role of the sbcC,D genes, and defines a new gene (rmuC) involved in
  DNA recombination.
  

  
  Here is a description of a PFAM that matches the gene:

  RmuC family
  
  This family contains several bacterial RmuC DNA recombination
  proteins. The function of the RMUC protein is unknown but it is
  suspected that it is either a structural protein that protects DNA
  against nuclease action, or is itself involved in DNA cleavage at the
  regions of DNA secondary structures.
  

An Example of a Hypothetical Protein Implicated in the TCA: E.coli Atomic Regulon 14

An Example Relating to Heme/Siroheme Biosynthesis: E.coli Atomic Regulon 16 [ON=748 OFF=115]

• The genes are from four distinct areas of the genome. Gene 232 is not in a cluster, 3727-3730 form a cluster, 3773-3776 form a cluster, and 3910-3912 form a cluster.
• Three of the four groups relate to Heme and Siroheme Biosynthesis.
• There are two peptidases -- one in the singleton group and one in a group containing a component of heme/siroheme biosynthesis.

A Membrane-bound Ni,Fe hydrogenase: E.coli Atomic Regulon 19 [ON=206 OFF=695]

Guessing the Substrate of a Transport System: Atomic Regulon 72 [ON=790 OFF=59]

Queuosine is a modified nucleoside that is present in certain tRNAs in
bacteria and eukaryotes.  Originally identified in E. coli,
queuosine was found to occupy the first anticodon position of tRNAs
for histidine, aspartic acid, asparagine and tyrosine.  The first
anticodon position pairs with the third "wobble" position in codons,
and queuosine improves accuracy of translation. Synthesis of
queuosine begins with GTP. In bacteria, two classes of riboswitch are
known to regulate genes that are involved in the synthesis or
transport of pre-queuosine1, a precursor to queuosine: PreQ1-I
riboswitches and PreQ1-II riboswitches.

A Hypothetical Connected to a Sulfur Relay Mediation Complex: E.coli Atomic Regulon 96 [ON=517 OFF=199]

Structure. 2006 Feb;14(2):357-66.

Structural basis for sulfur relay to RNA mediated by heterohexameric TusBCD complex

Numata T, Fukai S, Ikeuchi Y, Suzuki T, Nureki O

Abstract

Uridine at wobble position 34 of tRNA(Lys), tRNA(Glu), and tRNA(Gln)
is exclusively modified into 2-thiouridine (s2U), which is crucial for
both precise codon recognition and recognition by the cognate
aminoacyl-tRNA synthetases. Recent Escherichia coli genetic studies
revealed that the products of five novel genes, tusABCDE, function in
the s2U modification. Here, we solved the 2.15 angstroms crystal
structure of the E. coli TusBCD complex, a sulfur transfer mediator,
forming a heterohexamer composed of a dimer of the
heterotrimer. Structure-based sequence alignment suggested two
putative active site Cys residues, Cys79 (in TusC) and Cys78 (in
TusD), which are exposed on the hexameric complex. In vivo mutant
analyses revealed that only Cys78, in the TusD subunit, participates
in sulfur transfer during the s2U modification process. Since the
single Cys acts as a catalytic residue, we proposed that TusBCD
mediates sulfur relay via a putative persulfide state of the TusD
subunit.

and

Mol Cell. 2006 Jan 6;21(1):97-108

Mechanistic insights into sulfur relay by multiple sulfur mediators
involved in thiouridine biosynthesis at tRNA wobble positions.

Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T.

Abstract

The wobble bases of bacterial tRNAs responsible for NNR codons are
modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio
modification of mnm5s2U is required for accurate decoding and
essential for normal cell growth. We identified five genes yhhP, yheL,
yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE,
respectively) that are essential for 2-thiouridylation of mnm5s2U by a
systematic genome-wide screen ("ribonucleome analysis"). Efficient
2-thiouridine formation in vitro was reconstituted with recombinant
TusA, a TusBCD complex, TusE, and previously identified IscS and
MnmA. The desulfurase activity of IscS is stimulated by TusA
binding. IscS transfers the persulfide sulfur to TusA. TusE binds
TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE
also interacts with an MnmA-tRNA complex. This study revealed that
2-thiouridine formation proceeds through a complex sulfur-relay system
composed of multiple sulfur mediators that select and facilitate
specific sulfur flow to 2-thiouridine from various pathways of sulfur
trafficking.

YheO-like PAS domain:

This family contains various hypothetical bacterial proteins that are
similar to the E. coli protein YheO. Their function is unknown, but
are likely to be involved in signalling based on the presence of this
PAS domain.

A Component of a Complex: E.coli Atomic Regulon 109 [ON=125 OFF=755]

xanthine dehydrogenase accessory protein XdhC

Members of this protein family are the accessory protein XdhC for insertion of the 
molybdenum cofactor into the xanthine dehydrogenase large chain, XdhB, in bacteria. 
This protein is not part of the mature xanthine dehydrogenase. 
Xanthine dehydrogenase is an enzyme for purine catabolism, 
from other purines to xanthine to urate to further breakdown products.

The Glycine Cleavage System in Staph. aureus: Atomic Regulon 32 [ON=276 OFF=550]

JOURNAL OF BACTERIOLOGY, Oct. 1990, p. 6142-6144 Vol. 172, No. 10

The lpd Gene Product Functions as the L Protein in the
Escherichia coli Glycine Cleavage Enzyme System

PAULA S. STEIERT, LORRAINE T. STAUFFER, AND GEORGE V. STAUFFER

The enzyme serine hydroxymethyltransferase catalyzes the
conversion of serine to glycine and
5,10-methylenetetrahydrofolate and provides the major source of
one-carbon units. The oxidative cleavage of glycine to form
NH3, C02, and 5,10-methylenetetrahydrofolate provides a secondary
pathway for the biosynthesis of one-carbon units in mammalian and
bacterial systems. The glycine cleavage
(GCV) enzyme system has been described in Peptococcus
glycinophilus and consists of four protein components
designated P1, P2, P3, and P4. A GCV enzyme system that
can be induced by exogenous glycine has also been demonstrated
in Escherichia coli (9) and is located at minute 63 on
the linkage map (2). Although the system has not been well
characterized, at least one protein from E. coli interacts
catalytically with the P1 protein from P. glycinophilus in the
exchange of bicarbonate with 14C-labeled glycine, indicating
that the two systems may be similar (D. K. Ransom and
R. D. Sagers, Abstr. Annu. Meet. Am. Soc. Microbiol.
1974, P266, p. 189).

    "Gene "out of context": 

     In Chlamydia, Chlamidophyla, Streptococci, Bifidobacteria, Mycoplasma,
     Acinetobacter, etc - a very close homolog of  a Glycine cleavage
     system H protein is present, often - in close proximity of
     Lipoate-protein ligase A.  However, other Glycine cleavage
     system subunits are absent, the function of these proteins is
     unknown"

An Example Relating to Motility: Staph. aureus Atomic Regulon 2 [ON=229 OFF=12]

Pegs in Atomic Regulon 4 [ON=76 OFF=37]

Pegs in Atomic Regulon 6 [ON=65 OFF=48]

Pegs in Atomic Regulon 2 [ON=139 OFF=711]

Pegs in Atomic Regulon 3 [ON=390 OFF=451]

1. the two-component regulatory system implemented by peg.2391 and peg.2392 relate to "Nitrate and nitrite ammonification" (I am not even sure what that precisely means, but I think that it is straightforward to study the subsystem and literature to gain a detailed grasp), and
2. peg.2393 relates to "Nitrate and nitrite ammonification" or to the regulation of that process.

Pegs in Atomic Regulon 12 [ON=97 OFF=751]

• peg.400 has a hit against a domain described as "invasion associated secreted endopeptidase".
• peg.401 hits a domain described as "Intracellular trafficking and secretion".
• peg.402 hits a domain described by TIGR02746 as
  

  
  	type-IV secretion system protein TraC The protein family
          described here is common among the F, P and I-like type IV secretion
          systems. Gene symbols include TraC (F-type), TrbE/VirB4 (P-type) and
          TraU (I-type). The protein conyains the Walker A and B motifs and so
          is a putative nucleotide triphosphatase.
  

• peg.403 hits a domain described as
  

  	Antirestriction protein (ArdA)
  
          This family consists of several bacterial antirestriction (ArdA)
          proteins. ArdA functions in bacterial conjugation to allow an
          unmodified plasmid to evade restriction in the recipient bacterium and
          yet acquire cognate modification.
  

• a FIG annotator assigned to peg.404 the function "FIG086557: Conjugation related protein"